CD24-expressing cell and applications of the same

Abstract

To provide a cell including a cell expressing CD24, and related method of use and creation thereof.SOLUTION: Provided is an isolated cell including modification for increasing reduced expression of MHC class I and / or MHC class II human leukocyte antigens and expression of CD24 in a cell. In some embodiments, the cell includes reduced expression of MHC class I and MHC class II human leukocyte antigens.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 891,180, filed August 23, 2019, the disclosure of which is incorporated herein in its entirety. [Background technology]

[0002] Cancer and degenerative diseases pose a disproportionate threat to human health. Often associated with aging, these diseases result in the progressive deterioration of affected tissues and organs, ultimately resulting in disability and death of the affected subject. The promise of regenerative medicine lies in replacing diseased or lost cells with new, healthy cells. Over the past five years, a new paradigm in regenerative medicine has emerged: the use of human pluripotent stem cells (hPSCs) to generate any adult cell type for transplant into patients. In principle, hPSC-based cell therapy has the potential to treat most, if not all, degenerative diseases; however, the success of such treatments may be limited by the subject's immune response.

[0003] Strategies thought to overcome immune rejection include HLA matching (e.g., identical twins or umbilical cord blood banks), administering immunosuppressants to the subject, blocking antibodies, myelosuppression / mixed chimerism, HLA-matched stem cell repositories, and autologous stem cell therapy.

[0004] There remains a need for new approaches, compositions and methods to overcome immune rejection associated with cell replacement therapy. Summary of the Invention

[0005] In one aspect, provided herein is an isolated cell comprising reduced expression of MHC class I and / or MHC class II human leukocyte antigens and a modification to increase expression of CD24 on the cell. In some embodiments, the cell comprises reduced expression of MHC class I and MHC class II human leukocyte antigens.

[0006] In some embodiments, the cells further comprise a genetic modification that targets the CIITA gene with a rare-cutting endonuclease that selectively inactivates the CIITA gene. In some embodiments, the cells further comprise a modification to increase expression in the cells of a protein selected from the group consisting of CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9. In some embodiments, the cells further comprise a modification to increase expression of CD47 in the cells. In some embodiments, the cells further comprise a genetic modification that targets the B2M gene with a rare-cutting endonuclease that selectively inactivates the B2M gene. In some embodiments, the cells further comprise a genetic modification that targets the NLRC5 gene with a rare-cutting endonuclease that selectively inactivates the NLRC5 gene.

[0007] In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease. In some embodiments, the genetic modification targeting the CIITA gene with 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 CIITA gene. In some embodiments, In some embodiments, the genetic modification targeting the B2M gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the genetic modification targeting the NLRC5 gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene.

[0008] In some embodiments, the modification to increase CD24 expression comprises introducing into the cells an expression vector comprising a polynucleotide sequence encoding CD24. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having a sequence selected from the group consisting of SEQ ID NOs: 28-31.

[0009] In some embodiments, the modification to increase expression of one or more selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 comprises introducing into the cell an expression vector comprising a polynucleotide sequence encoding one or more selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35. In some embodiments, the modification to increase expression of CD47 comprises introducing into the cell an expression vector comprising a polynucleotide sequence encoding CD47.

[0010] In some embodiments, the expression vector for increasing expression of any of the described polypeptides is an inducible expression vector, hi some embodiments, the expression vector is a viral vector.

[0011] In some embodiments, the modification to increase CD24 expression comprises introducing a polynucleotide sequence encoding CD24 into a selected locus of the cell. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having a sequence selected from the group consisting of SEQ ID NOs: 28-31.

[0012] In some embodiments, the modification to increase expression of a polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 comprises introducing into a selected locus of the cell a polynucleotide sequence encoding a polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35. In some embodiments, the modification to increase expression of CD47 comprises introducing into a selected locus of the cell a polynucleotide sequence encoding CD47. In some embodiments, the selected genetic locus for a polynucleotide sequence encoding CD24 and / or the selected genetic locus for a polynucleotide sequence encoding a polynucleotide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35. The child locus is a safe harbor locus. In some embodiments, the safe harbor is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

[0013] In some embodiments, the cell further comprises an inducible suicide switch.

[0014] In some embodiments, the cells are selected from the group consisting of stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

[0015] In some aspects, provided herein are methods of preparing cells comprising CD24 (e.g., a CD24 polypeptide), the methods comprising introducing into cells an expression vector comprising a polynucleotide sequence encoding CD24, thereby producing cells comprising CD24. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having a sequence selected from the group consisting of SEQ ID NOs: 28-31.

[0016] In some embodiments, the cells comprising CD24 further comprise a genetic modification targeting the CIITA gene, comprising a rare-cutting endonuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease, for targeting the CIITA gene. In some embodiments, the genetic modification comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid for specifically targeting the CIITA gene.

[0017] In some embodiments, the expression vector comprising a polynucleotide sequence encoding CD24 is an inducible expression vector. In some embodiments, the expression vector is a viral vector.

[0018] In some embodiments, the cells comprising CD24 further comprise a second expression vector comprising a polynucleotide sequence encoding one selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35. In some embodiments, the second expression vector comprises a polynucleotide sequence encoding CD47. In some embodiments, the second expression vector is an inducible expression vector. In some embodiments, the second expression vector is a viral vector.

[0019] In some embodiments, the cells comprising CD24 further comprise a genetic modification targeting the B2M gene, comprising a rare-cutting endonuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease, for specifically targeting the B2M gene. In some cases, the genetic modification comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid for specifically targeting the B2M gene.

[0020] In some embodiments, the CD24-containing cells are transfected with a rare nuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease to specifically target the NLRC5 gene. The method further includes a genetic modification that targets the NLRC5 gene, including a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid for specifically targeting the NLRC5 gene.

[0021] In some embodiments, the cell is selected from the group consisting of stem cells, differentiated cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

[0022] Provided herein is a method for preparing hypoimmunogenic stem cells, which involves introducing a polynucleotide sequence encoding CD24 into a selected locus in a stem cell, thereby producing hypoimmunogenic stem cells.

[0023] In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having at least 90% or at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28 to 31. In some embodiments, the polynucleotide sequence encoding CD24 is a nucleotide sequence that encodes a polypeptide sequence having a sequence selected from the group consisting of SEQ ID NOs: 28 to 31.

[0024] In some embodiments, the method further comprises generating a genetic modification targeting the CIITA gene in the stem cell, comprising introducing a rare-cutting endonuclease into the stem cell that selectively inactivates the CIITA gene, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease. In some embodiments, introducing the rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid for specifically targeting the CIITA gene.

[0025] In some embodiments, the selected locus for a polynucleotide sequence encoding CD24 is a safe harbor locus, hi some embodiments, the safe harbor locus for a polynucleotide sequence encoding CD24 is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

[0026] In some embodiments, the method for preparing hypoimmunogenic stem cells further comprises introducing a polynucleotide sequence encoding a polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 into a selected locus of the stem cells. In certain embodiments, the method further comprises introducing a polynucleotide sequence encoding CD47 into a selected locus of the stem cells. In some embodiments, the selected locus is a safe harbor locus. In some embodiments, the safe harbor locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

[0027] In some embodiments, the method for preparing hypoimmunogenic stem cells further comprises generating a genetic modification targeting the B2M gene in the stem cells, comprising introducing into the stem cells a rare-cutting endonuclease that selectively inactivates the B2M gene, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, and the like. In some embodiments, the introduction of the rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid to specifically target the B2M gene.

[0028] In some embodiments, the method for preparing hypoimmunogenic stem cells further comprises generating a genetic modification targeting the NLRC5 gene in the stem cell, comprising introducing into the stem cell a rare-cutting endonuclease that selectively inactivates the NLRC5 gene, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease. In some embodiments, introducing the rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid to specifically target the NLRC5 gene.

[0029] In some embodiments, the method for preparing hypoimmunogenic stem cells further comprises introducing into the stem cells an expression vector comprising an inducible suicide switch.

[0030] In some aspects, provided is a method of preparing differentiated hypoimmunogenic cells, comprising culturing hypoimmunogenic stem cells prepared according to any of the methods disclosed herein under differentiation conditions, thereby preparing differentiated hypoimmunogenic cells.

[0031] In some embodiments, the differentiation conditions are suitable for differentiation of stem cells into a cell type selected from the group consisting of cardiac cells, neural cells, endothelial cells, T cells, pancreatic islet cells, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

[0032] In some aspects, provided are methods of treating a patient in need of cell therapy comprising administering a population of differentiated, hypoimmunogenic cells prepared according to the methods disclosed herein.

[0033] Provided herein are cells that express CD24 and have reduced expression of MHC class I human leukocyte antigens.

[0034] Provided herein are cells that express CD24 and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0035] Provided herein are cells that do not express CIITA, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0036] Provided herein are cells that do not express B2M, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0037] Provided herein are cells that express NLRC5, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0038] Provided herein are cells that express CD24 and at least one selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens. do.

[0039] Provided herein are cells that express CD24 and CD47 and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0040] Provided herein are cells that do not express CIITA, express CD24 and at least one polypeptide selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0041] Provided herein are cells that do not express CIITA, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0042] Provided herein are cells that do not express CIITA and B2M, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0043] Provided herein are cells that do not express CIITA and B2M, express CD24 and at least one polypeptide selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0044] Provided herein are cells that do not express CIITA and B2M, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0045] Provided herein are cells that do not express CIITA and NLRC5, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0046] Provided herein are cells that do not express CIITA and NLRC5, express CD24 and at least one polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0047] Provided herein are cells that do not express CIITA and NLRC5, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0048] Provided herein are cells that do not express CIITA, B2M, and NLRC5, but express CD24 and at least one polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0049] Provided herein are medicaments that do not express CIITA, B2M, and NLRC5 and that are CD4+. 24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

[0050] In some embodiments, any one of the above cells is selected from the group consisting of stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

[0051] Also provided herein are differentiated cells produced from any of the pluripotent stem cells or induced pluripotent stem cells described herein by culturing under differentiation conditions to produce differentiated cells selected from the group consisting of cardiac cells, neural cells, endothelial cells, T cells, pancreatic islet cells, retinal pigment epithelial (RPE) cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

[0052] In one aspect of the present disclosure, provided herein is an isolated stem cell comprising an exogenous CD24 polypeptide. In some embodiments, the cell expresses a nucleotide (e.g., polynucleotide) sequence encoding a CD24 polypeptide having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the cell expresses a nucleotide sequence encoding a CD24 polypeptide having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the cell expresses a nucleotide sequence encoding a CD24 polypeptide selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0053] In some embodiments, the isolated cells have reduced expression of MHC class I human leukocyte antigen. In other embodiments, the cells have reduced expression of MHC II human leukocyte antigen. In still other embodiments, the cells have reduced expression of MHC class I and MHC II human leukocyte antigen. In some embodiments, the cells have reduced expression of CIITA. In certain embodiments, the cells have reduced expression of B2M. In certain embodiments, the cells have reduced expression of NLRC5.

[0054] In some embodiments, the isolated cell further comprises a genomic modification targeting CIITA to reduce its expression. In some embodiments, the genomic modification comprises a rare-cutting endonuclease. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. In certain embodiments, the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein targeting CIITA. Optionally, the cell further comprises at least one guide ribonucleic acid sequence recognized by the Cas protein targeting CIITA. In some embodiments, the at least one guide ribonucleic acid sequence for targeting CIITA is selected from the group consisting of SEQ ID NOs: 5184-36352 of WO2016 / 183041, the disclosure of which, including its sequence listing, is incorporated by reference in its entirety.

[0055] In other embodiments, the isolated cell further comprises a genomic modification that targets B2M to reduce expression of B2M. In some embodiments, the genomic modification comprises a rare-cutting endonuclease. In some embodiments, the rare-cutting endonuclease is a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, or a nucleotide sequence encoding a nucleotide sequence. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein or a polynucleotide encoding a Cas protein that targets B2M. In some embodiments, the cell further comprises at least one guide ribonucleic acid sequence recognized by the Cas protein that targets B2M. In some embodiments, the at least one guide ribonucleic acid sequence for targeting B2M is selected from the group consisting of SEQ ID NOs: 81240-85644 of WO2016 / 183041, the disclosure of which, including the sequence listing, is incorporated by reference in its entirety.

[0056] In some embodiments, the isolated cell further comprises a genomic modification that targets NLRC5 to reduce its expression. In some embodiments, the genomic modification comprises a rare-cutting endonuclease. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. In some embodiments, the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein that targets NLRC5. Optionally, the cell further comprises at least one guide ribonucleic acid sequence recognized by the Cas protein that targets NLRC5. In some embodiments, the at least one guide ribonucleic acid sequence for targeting NLRC5 is selected from the group consisting of SEQ ID NOs: 36353-81239 of WO2016 / 183041, the disclosure of which, including the sequence listing, is incorporated by reference in its entirety.

[0057] In some embodiments, the isolated cells further comprise a gene expression modification to reduce the expression of CIITA. In certain embodiments, the cells further comprise a gene expression modification to reduce the expression of B2M. In other embodiments, the cells further comprise a gene expression modification to reduce the expression of NLRC5. In some embodiments, the gene expression modification comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule.

[0058] In some embodiments, the isolated cells further comprise an exogenous immune modulator selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35. In other embodiments, the cells further comprise one or more exogenous immune modulators selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35.

[0059] In some embodiments, the isolated cells outlined herein are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

[0060] In some embodiments, provided herein are isolated cells generated from any of the stem cells described herein under differentiation conditions.

[0061] In some embodiments, provided herein are isolated cells that are hypoimmunogenic, eg, hypoimmunogenic to a patient upon administration.

[0062] In one aspect, provided herein is a method of preparing stem cells comprising an exogenous CD24 polypeptide, the method comprising introducing an expression vector comprising a nucleotide sequence encoding a CD24 polypeptide having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the CD24 polypeptide sequence is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32. In another aspect, provided herein is a method of preparing stem cells comprising an exogenous CD24 polypeptide, the method comprising introducing an expression vector comprising a nucleotide sequence encoding a CD24 polypeptide having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the CD24 polypeptide sequence is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0063] In some embodiments, the expression vector of the exogenous CD24 polynucleotide is an inducible expression vector. In some embodiments, the expression vector is a viral vector. In certain embodiments, the expression vector specifically targets a safe harbor locus. In certain embodiments, the safe harbor locus is the AAVS1 locus.

[0064] In some embodiments, the method for preparing stem cells further comprises introducing into the cells a rare-cutting endonuclease that selectively inactivates the CIITA gene. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. Optionally, the method also comprises introducing at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOs: 5184-36352 of WO2016 / 183041, the disclosure of which, including the sequence listing, is incorporated by reference in its entirety.

[0065] In some embodiments, the method further comprises introducing into the cell a rare-cutting endonuclease that selectively inactivates the B2M gene. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. Optionally, the method also comprises introducing at least one guide ribonucleic acid sequence for specifically targeting the B2M gene, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein. In some embodiments, 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 WO2016 / 183041, the disclosure of which, including the sequence listing, is incorporated by reference in its entirety.

[0066] In some embodiments, the method further comprises introducing into the cell a rare-cutting endonuclease that selectively inactivates the NLRC5 gene. In some embodiments, the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. Optionally, the method also comprises introducing at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein. In some embodiments, 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 WO2016 / 183041, the disclosure of which, including the sequence listing, is incorporated by reference in its entirety.

[0067] In some embodiments, the method further comprises introducing a gene expression-modifying molecule into the cell to reduce expression of CIITA, wherein the gene expression-modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets CIITA. In some embodiments, the method further comprises introducing a gene expression-modifying molecule into the cell to reduce expression of B2M, wherein the gene expression-modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets B2M. In some embodiments, the method further comprises introducing a gene expression-modifying molecule into the cell to reduce expression of NLRC5, wherein the gene expression-modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets NLRC5.

[0068] In some embodiments, the method further comprises introducing an expression vector comprising a nucleotide sequence encoding a tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35.

[0069] In some embodiments, the method further comprises introducing at least two expression vectors, wherein a first expression vector comprises a first nucleotide sequence encoding a first tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35, and a second expression vector comprises a second nucleotide sequence encoding a different tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35.

[0070] In some embodiments, the expression vector, the first expression vector, and / or the second expression vector described herein is an inducible expression vector. In some embodiments, the expression vector, the first expression vector, and / or the second expression vector described herein is a viral vector. In some embodiments, the expression vector, the first expression vector, and / or the second expression vector described herein specifically targets a safe harbor locus. In some cases, the safe harbor locus is the AAVS1 locus.

[0071] In some embodiments, the method further comprises introducing into the stem cell an expression vector comprising an inducible suicide switch.

[0072] In some embodiments, the stem cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, and adult stem cells.

[0073] In some embodiments, the stem cells have reduced expression of MHC class I human leukocyte antigens compared to unmodified stem cells. In some embodiments, the stem cells have reduced expression of MHC II human leukocyte antigens compared to unmodified stem cells. In some embodiments, the stem cells have reduced expression of MHC class I and MHC II human leukocyte antigens compared to unmodified stem cells.

[0074] In another aspect, provided herein is a method for preparing differentiated cells, comprising culturing any one of the stem cells described herein or any one of the stem cells prepared according to the methods outlined herein under differentiation conditions, thereby preparing differentiated cells. In some embodiments, the differentiation conditions are such that the stem cells are differentiated into cardiac cells, hepatocytes, The cells are suitable for differentiation into cell types selected from the group consisting of kidney cells, pancreatic cells, neural cells, immune cells, mesenchymal cells, and endothelial cells.

[0075] In another aspect, provided herein is a method of treating a patient in need of a cell-based therapy, such as, but not limited to, cell replacement therapy, comprising administering a population of differentiated cells prepared according to any of the methods outlined herein.

[0076] Provided herein are stem cells that express exogenous CD24 polypeptide and reduced expression levels of MHC class I human leukocyte antigen. Provided herein are stem cells that express exogenous CD24 polypeptide and reduced expression levels of MHC class II human leukocyte antigen. Provided herein are stem cells that express exogenous CD24 polypeptide and reduced expression levels of MHC class I and class II human leukocyte antigen.

[0077] Provided herein is a stem cell that expresses exogenous CD24 polypeptide and a reduced expression level of CIITA. Provided herein is a stem cell that expresses exogenous CD24 polypeptide and a reduced expression level of B2M. Provided herein is a stem cell that expresses exogenous CD24 polypeptide and a reduced expression level of NLRC5. Provided herein is a stem cell that expresses exogenous CD24 polypeptide and a reduced expression level of CIITA, B2M, NLRC5, and combinations thereof.

[0078] Provided herein are stem cells that express one or more tolerogenic factors selected from the group consisting of exogenous CD24 polypeptide, HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35. Provided herein are stem cells that express exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of CIITA. Provided herein are stem cells that express exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of B2M. Provided herein are stem cells that express an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of NLRC5. Provided herein are stem cells that express an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

[0079] Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide and reduced expression levels of MHC class I human leukocyte antigen. Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide and reduced expression levels of MHC class II human leukocyte antigen. Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide and reduced expression levels of MHC class I and class II human leukocyte antigen.

[0080] Provided herein are differentiated cells generated from stem cells that express exogenous CD24 polypeptide and reduced expression levels of CIITA. Provided herein are differentiated cells generated from stem cells that express exogenous CD24 polypeptide and reduced expression levels of B2M. Provided herein are differentiated cells generated from stem cells that express exogenous CD24 polypeptide and reduced expression levels of NLRC5. Provided herein are differentiated cells generated from stem cells that express exogenous CD24 polypeptide and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

[0081] Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide and one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35. Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of CIITA. Provided herein are differentiated cells generated from stem cells expressing exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of B2M. Provided herein are differentiated cells generated from stem cells that express an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of NLRC5. Provided herein are differentiated cells generated from stem cells that express an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

[0082] In some embodiments, the stem cells of the present invention are hypoimmunogenic, e.g., hypoimmunogenic to a patient upon administration, hi some embodiments, the differentiated cells of the present invention are hypoimmunogenic, e.g., hypoimmunogenic to a patient upon administration.

[0083] In some embodiments, provided herein are methods of treating a patient in need of cell therapy (optionally, cell replacement therapy), comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells that express reduced expression levels of MHC class I human leukocyte antigen.

[0084] In some embodiments, provided herein are methods of treating a patient in need of cell therapy (optionally, cell replacement therapy), comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells that express reduced expression levels of MHC class II human leukocyte antigen.

[0085] In some embodiments, provided herein are methods of treating a patient in need of cell therapy (optionally, cell replacement therapy), comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells that express reduced expression levels of MHC class I and class II human leukocyte antigens.

[0086] In some embodiments, provided herein are methods of treating a patient in need of cell therapy (optionally, cell replacement therapy), comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells that have reduced expression levels of CIITA. Provided herein are methods of treating a patient in need of cell therapy, comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells expressing reduced expression levels of B2M. In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells expressing reduced expression levels of NLRC5. In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering an exogenous CD24 polypeptide and a population of differentiated cells comprising differentiated cells generated from stem cells expressing reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

[0087] In some embodiments, provided herein are methods of treating a patient in need of cell therapy (optionally, cell replacement therapy), comprising administering a population of differentiated cells comprising differentiated cells generated from stem cells that express exogenous CD24 polypeptide and one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35. In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering a population of differentiated cells comprising differentiated cells generated from stem cells that express exogenous CD24 polypeptide and one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of CIITA. In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering a population of differentiated cells comprising differentiated cells generated from stem cells that express exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of B2M. In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering a population of differentiated cells comprising differentiated cells generated from stem cells that express exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of NLRC5.In some embodiments, provided herein are methods of treating a patient in need of cell therapy, comprising administering a population of differentiated cells comprising differentiated cells generated from stem cells that express exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

[0088] A detailed description of the hypoimmunogenic cells, methods for their production, and methods for their use is set forth in WO2016 / 183041, filed May 9, 2015, WO2018 / 132783, filed January 14, 2018, and WO2018 / 175390, filed March 20, 2018, the disclosures of which, including sequence listings and figures, are incorporated herein by reference in their entireties.

[0089] Other objects, advantages and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0090] [Figures 1A-1H] The sequences of DUX4 polynucleotide and DUX4, CD47, and CD24 polypeptides are shown as SEQ ID NOs: 1 to 33. DETAILED DESCRIPTION OF THE INVENTION

[0091] I. Introduction Genome editing and the generation of induced pluripotent stem cells (iPSCs), followed by differentiation of such iPSCs, remains a costly, time-consuming, and highly variable process with regard to pluripotency, epigenetic state, differentiation potential, and genomic stability. Furthermore, changes that occur during genome editing and long-term culture have been found to trigger adaptive immune responses, resulting in immune rejection of even autologous stem cell-derived transplants or explants. To overcome the problem of immune rejection in subjects of stem cell-derived transplants, the present inventors have developed and disclosed herein hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells, hypoimmunogenic differentiated cells, hypoimmunogenic primary T cells, etc.) that represent a viable source of transplantable cell types. Such CD24-expressing cells are protected from adaptive and innate immune rejection upon administration to recipient subjects. Advantageously, the cells disclosed herein are not rejected by the recipient subject's immune system, regardless of the subject's genetic makeup. Such cells are protected from adaptive and innate immune rejection upon administration to recipient subjects.

[0092] In some embodiments, the CD24 expressing hypoimmunogenic cells outlined herein are not subject to natural immune cell rejection. In some cases, hypoimmunogenic cells are not susceptible to NK cell-mediated lysis. In some cases, hypoimmunogenic cells are not susceptible to macrophage phagocytosis. In some embodiments, hypoimmunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into recipient subjects with little or no need for immunosuppressants. Such hypoimmunogenic cells retain cell-specific properties and characteristics upon transplantation.

[0093] In some embodiments, provided herein are stem cells or differentiated derivatives thereof that avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced from the stem cells outlined herein avoid immune rejection when administered (e.g., transplanted or grafted) to an MHC-mismatched allogeneic recipient. In other words, stem cells and differentiated cells derived from such stem cells (including their progeny) are hypoimmunogenic. In some embodiments, the hypoimmunogenic stem cells outlined herein have reduced immunogenicity (e.g., at least 2.5% to 99% less immunogenicity) compared to wild-type stem cells. In some cases, the hypoimmunogenic stem cells lack immunogenicity compared to wild-type stem cells. Stem cells or differentiated derivatives thereof are suitable as universal donor cells for transplantation or engraftment into recipient patients. In some embodiments, such cells are non-immunogenic to the patient. In some embodiments, provided herein are stem cells with reduced immunogenicity. Such stem cells retain the potential and differentiation capacity of pluripotent stem cells.

[0094] The provided methods are useful for inactivating or eliminating MHC class I and / or MHC class II expression in cells, such as, but not limited to, pluripotent stem cells. In some embodiments, genome editing techniques utilizing rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of important immune genes in human stem cells (e.g., by deleting the genomic DNA of important immune genes). In certain embodiments, genome editing or other gene regulation techniques are used to insert immune tolerance-inducing factors into human cells, rendering them and differentiated cells prepared therefrom hypoimmunogenic. In this way, hypoimmunogenic cells have reduced or eliminated expression of MHC I and MHC II expression. In some embodiments, the cells are non-immunogenic in a recipient subject (e.g., by inhibiting an immune response). (Do not induce

[0095] Genome editing technology allows for double-stranded DNA breaks at the desired locus. These controlled double-stranded breaks promote homologous recombination at the specific locus. This process focuses on targeting specific sequences in nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to those sequences and induce double-stranded breaks in the nucleic acid molecule. The double-stranded breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0096] The practice of certain embodiments will employ, unless otherwise indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for purposes of illustration. Such techniques are explained fully in the literature, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2001); 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992), Transcription and Translation (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide See monographs in journals such as Molecular Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0097] II. Definition When used herein to characterize cells, the term "low immunogenicity" generally refers to a reduced tendency for immune rejection by a subject into which such cells are transplanted. For example, compared to unaltered or unmodified wild-type cells, such low immunogenic cells are about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or less less likely to be immune rejected by a subject into which such cells are transplanted. In some aspects, genome editing technology is used to regulate the expression of MHC I and MHC II genes, thus generating low immunogenic cells. In some embodiments, the low immunogenic cells avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced from the low immunogenic stem cells outlined herein are administered to MHC-mismatched allogeneic recipients. Avoid immune rejection when (e.g., transplanted or grafted). In some embodiments, the hypoimmunogenic cells are protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection.

[0098] The low immunogenicity of cells can be determined by assessing the immunogenicity of the cells, such as their ability to induce adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, immune response assays measure the effect of the low immunogenic cells on T cell proliferation, T cell activation, T cell killing, NK cell proliferation, NK cell activation, and macrophage activation. In some cases, the low immunogenic cells and their derivatives undergo reduced killing by T cells and / or NK cells upon administration to a subject. In some cases, the cells and their derivatives exhibit reduced macrophage phagocytosis compared to unmodified or wild-type cells. In some embodiments, the low immunogenic cells induce a reduced or diminished immune response in a recipient subject compared to corresponding unmodified wild-type cells. In some embodiments, the low immunogenic cells are non-immunogenic or unable to induce an immune response in a recipient subject.

[0099] As used herein, "immunosuppressive factors" or "immunomodulatory factors" or "tolerogenic factors" include hypoimmune factors, complement inhibitors, and other factors that modulate or influence the ability of cells to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment.

[0100] As used herein, "immune signaling factor" refers to a molecule, protein, peptide, etc. that activates an immune signaling pathway, as the case may be.

[0101] As used herein, a "safe harbor locus" refers to a locus that allows safe expression of a transgene or exogenous gene. Exemplary "safe harbor" loci include the CCR5 gene, the CXCR4 gene, the PPP1R12C (also known as AAVS1) gene, the albumin gene, the SHS231 locus, the CLYBL gene, and the Rosa gene (e.g., ROSA26).

[0102] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of that gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0103] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that have been modified by processes such as capping, polyadenylation, methylation, and editing, and proteins that have been modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0104] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation can also be complete, i.e., gene expression is completely inactivated or reduced to wild-type levels. Alternatively, it may be partial, where gene expression is partially reduced or partially activated to some fraction of wild-type levels.

[0105] The terms "operatively linked" or "operably linked" are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) where the components are positioned to allow for the normal function of both components and the potential for at least one of the components to mediate a function exerted by at least one of the other components. Illustratively, a transcriptional regulatory sequence such as a promoter is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to the coding sequence. For example, an enhancer is a transcriptional regulatory sequence operably linked to a coding sequence even if the enhancer and coding sequence are not contiguous.

[0106] A "vector" or "construct" can transfer a gene sequence into a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct that can direct the expression of a gene of interest and transfer a gene sequence into a target cell. Thus, the term includes cloning, expression vehicles, and integration vectors. Methods for introducing a vector or construct into a cell are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle gun technology, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0107] As used herein, "pluripotent stem cells" have the potential to differentiate into any of the three germ layers: endoderm (e.g., gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue), or ectoderm (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cells" also encompasses "induced pluripotent stem cells," or "iPSCs," or a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from cells that are not pluripotent. In other words, pluripotent stem cells can be the direct or indirect progeny of a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be generated by inducing the expression of specific regulatory genes or by the exogenous application of specific proteins. Methods for deriving iPS cells are known in the art and are described further below. (For example, Zhou et al., Stem Cells 27(11):2667-74(2009), Huangfu et al., Nature Biotechnol. 26(7):795 (2008), Woltjen et al., Nature 458(7239):766-770 (2009), and Zhou et al., Cell Stem Cell 8:381-384 (2009), each of which is incorporated herein by reference in its entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSCs" refers to human induced pluripotent stem cells.

[0108] The "HLA" or "human leukocyte antigen" complex is a gene complex that encodes human major histocompatibility complex (MHC) proteins. These cell surface proteins that make up the HLA complex are involved in regulating the immune response to antigens. Humans have two MHC classes, class I and class II, "HLA-I" and "HLA-II." HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, which are involved in intracellular The HLA-I complex presents peptides from the HLA-I complex, and antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). The HLA-I protein is associated with beta-2 microglobulin (B2M). HLA-II contains five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which extracellularly present antigens to T lymphocytes, which stimulate CD4+ cells (also known as T helper cells). It should be understood that the use of either "MHC" or "HLA" is not meant to be limiting, as it depends on whether the genes are human (HLA) or mouse (MHC). Therefore, these terms may be used interchangeably herein as they relate to mammalian cells.

[0109] As used herein, the terms "graft," "administration," "introduction," "implanting," and "transplanting," as well as grammatical variations thereof, are used interchangeably in the context of placing cells (e.g., cells described herein) into a subject by a method or route that results in at least partial localization of the introduced cells at the desired site. Cells can be directly implanted at the desired site or can be administered by any suitable route that results in delivery to the desired location in the subject where at least a portion of the transplanted cells or cellular components remain viable. The survival period of cells after administration to a subject can be as short as a few hours, e.g., 24 hours to several days, or as long as several years. In some embodiments, cells can also be administered (e.g., injected) at a location other than the desired site, e.g., intracerebrally or subcutaneously, e.g., in a capsule, to maintain the transplanted cells at the transplanted location and prevent migration of the transplanted cells.

[0110] The terms "treat," "treating," "treatment," and the like, as applied to isolated cells, include subjecting the cells to any type of process or condition, or performing any type of operation or procedure on the cells. When applied to a subject, these terms refer to administering to an individual a cell or population of cells in which a target polynucleotide sequence (e.g., B2M) has been modified ex vivo according to the methods described herein. The individual is typically ill or injured, or at increased risk of developing a disease compared to the average member of the population, and in need of such care, attention, or management.

[0111] As used herein, the terms "treating" and "treatment" refer to administering to a subject an effective amount of cells having a target polynucleotide sequence modified ex vivo according to the methods described herein, such that the subject experiences at least one symptom or disease amelioration, e.g., a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of disease, a stable (i.e., not worsening) state of disease, a delay or slowing of disease progression, an improvement or alleviation of disease symptoms, and remission (partial or total). Treating also includes prolonging survival compared to expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment may improve disease symptoms, but may not completely cure the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is slowed or halted. "Treatment" also means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with a disorder associated with expression of the polynucleotide sequence as well as those who are likely to develop such a disorder due to genetic susceptibility or other factors.

[0112] "Treatment," "prevention," or "amelioration" of a disease or disorder means delaying or preventing the onset of such disease or disorder, or preventing the progression or worsening of the conditions associated with such disease or disorder. or to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a disease or disorder. In one embodiment, the 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%.

[0113] The term "cancer," as used herein, is defined as an excessive proliferation of cells whose unique characteristics (e.g., loss of normal control) result in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the methods of the present invention, cancer includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, nose, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma. The tumor may be any cancer, including any of the following: hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue of a malignant type, and does not include benign type tissue, unless otherwise specified.

[0114] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which infection persists. Such diseases can include hepatitis (A, B, or C), herpes viruses (such as VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples can include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents can be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0115] The term "autoimmune disease" refers to any disease or disorder in which a subject launches a destructive immune response against its own tissues. Autoimmune disorders can affect almost any organ system in a subject (e.g., human), including but not limited to diseases of the nervous system, gastrointestinal system, and endocrine system, as well as skin and other connective tissues, eyes, blood, and blood vessels. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0116] In additional or alternative embodiments, the present invention contemplates that any method available to those skilled in the art can be used to modify target polynucleotide sequences, for example, by using nuclease systems, such as TAL effector nuclease (TALEN) or zinc finger nuclease (ZFN) systems.Although examples of methods using CRISPR / Cas (for example, Cas9 and Cpf1) and TALEN are described in detail herein, it should be understood that the present invention is not limited to the use of these methods / systems.Other targeting methods known to those skilled in the art can be used herein to reduce or eliminate expression in target cells, such as B2M.

[0117] The methods of the present invention can be used to alter a target polynucleotide sequence in a cell. The present invention contemplates altering a target polynucleotide sequence in a cell for any purpose. FIG. 1 illustrates a target polynucleotide sequence in a cell that is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell that has a resulting genotype that differs from its original genotype. In some cases, a "mutant cell" exhibits a mutant phenotype, e.g., when a normally functioning gene is altered using the CRISPR / Cas system of the present invention. In other instances, a "mutant cell" exhibits a wild-type phenotype, e.g., when the CRISPR / Cas system of the present invention is used to correct a mutant genotype. In some embodiments, a target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, a target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0118] In some embodiments, the alteration is an indel. As used herein, "indel" refers to a mutation resulting from insertion, deletion, or a combination thereof. As understood by those skilled in the art, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The CRISPR / Cas system of the present invention can be used to induce indels of any length or point mutations in target polynucleotide sequences.

[0119] As used herein, "knockout" includes deleting all or part of a target polynucleotide sequence in a manner that disrupts the function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by introducing an indel into the target polynucleotide sequence of a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Those skilled in the art will readily understand how to use the CRISPR / Cas system of the present invention to knock out a target polynucleotide sequence or part thereof based on the details described herein.

[0120] In some embodiments, the alteration results in knockout of the target polynucleotide sequence or a portion thereof. Knocking out a target polynucleotide sequence or a portion thereof using the CRISPR / Cas system of the present invention can be useful for various applications. For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing disorders associated with the expression of the target polynucleotide sequence (for example, by knocking out a mutant allele in a cell ex vivo and introducing the knocked-out mutant allele into a subject).

[0121] As used herein, "knock-in" refers to the process of adding a genetic function to a host cell. This results in increased levels of the knocked-in gene product, such as RNA or the encoded protein. As will be understood by those skilled in the art, this can be achieved in several ways, including adding one or more additional copies of the gene to the host cell or modifying the regulatory components of the endogenous gene to increase protein expression. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0122] In some embodiments, the changes or modifications described herein result in a decrease in expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in a decrease in expression of a target or selected polypeptide sequence. bring about.

[0123] In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polypeptide sequence.

[0124] The terms "decrease," "reduced," "reduction," and "decrease" are all used herein generally to mean a statistically significant decrease. For the avoidance of doubt, however, "decrease," "reduced," "reduction," and "decrease" mean a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e., a level of absence compared to a reference sample), or any decrease between 10-100% compared to a reference level.

[0125] The terms "increased," "increase," "enhance," or "activate" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," "enhance," or "activate" mean an increase of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0126] As used herein, the term "exogenous" is intended to mean that the referenced molecule or polypeptide is introduced into a cell of interest. A polypeptide can be introduced, for example, by introducing an encoding nucleic acid into the cell's genetic material, such as by chromosomal integration, or as non-chromosomal genetic material, such as a plasmid or expression vector. Thus, the term "exogenous" used in reference to the expression of an encoding nucleic acid refers to the introduction of an expressible form of the encoding nucleic acid into a cell. An "exogenous" molecule is a molecule, construct, factor, etc. that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods. Its "normal presence in a cell" is determined with respect to the cell's particular developmental stage and environmental conditions. Thus, for example, a molecule that is present only during neuronal embryonic development is an exogenous molecule with respect to an adult neuronal cell. An exogenous molecule can include, for example, a functioning version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0127] The exogenous molecule or factor may be, inter alia, a small molecule, such as one produced by combinatorial chemical processes, or a macromolecule, such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, and can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include those that can form duplexes as well as triplexes. See, e.g., U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include DNA-binding proteins, transcription factors, These include, but are not limited to, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0128] The term "endogenous" refers to a referenced molecule or polypeptide that is present in a cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0129] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a certain percentage of the same nucleotides or amino acid residues when compared and aligned to their closest counterparts, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent "identity" can be over a region of the sequences being compared, such as a functional domain, or over the entire length of the two sequences being compared. In sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0130] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by visual inspection (see generally Ausubel et al., infra).

[0131] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0132] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, e.g., a human, from which cells can be obtained and / or to which treatment, including prophylactic treatment, with the cells described herein is provided. For treatment of those infections, conditions, or pathologies specific to a particular animal, such as a human subject, the term subject refers to that particular animal. "Non-human animals" and "non-human mammals," used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., a dog, cat, or horse. , or productive mammals, such as cattle, sheep, pigs, etc.

[0133] It should be noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prelude to the use of exclusive terms such as "only," "only," and the like in connection with the recitation of claim elements or the use of "negative" limitations. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises distinct components and features that may be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0134] In describing the present invention, the following terms will be used and are defined as set forth below.

[0135] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are described herein by numerical values ​​preceded by the term "about." The term "about" is used herein to literally support the exact number that follows and a number that is close to or approximately the number that follows the term. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context presented, provides a substantial equivalent to the specifically recited number.

[0137] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter related to which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the inventions described herein are not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0138] III. Detailed Description of the Embodiments A. Low immunogenic cells Provided herein are modifications to increase expression of CD24 and MHC class II antibodies. In some embodiments, the cells comprise one or more target polynucleotide sequence modifications that regulate expression of MHC class I and / or MHC class II human leukocyte antigens. In some embodiments, the cells comprise an exogenous CD24 polypeptide. In some embodiments, the cells also comprise modifications to increase expression of one or more polypeptides selected from the group consisting of CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb95. In some embodiments, the described cells are those expressing exogenous CD24 and CD47 polypeptides, exogenous CD24 and DUX4 polypeptides, exogenous CD24 and CD27 polypeptides, exogenous CD24 and CD35 polypeptides, exogenous CD24 and CD46 polypeptides, exogenous CD24 and CD55 polypeptides, exogenous CD24 and CD59 polypeptides, exogenous CD24 and CD200 polypeptides, exogenous CD24 and HLA-C polypeptides, exogenous CD24 and HLA-E polypeptides, exogenous CD24 and HLA-E heavy chain polypeptides. exogenous CD24 and HLA-G polypeptides, exogenous CD24 and PD-L1 polypeptides, exogenous CD24 and IDO1 polypeptides, exogenous CD24 and CTLA4 polypeptides, exogenous CD24 and C1-inhibitor polypeptides, exogenous CD24 and IL-10 polypeptides, exogenous CD24 and IL-35 polypeptides, exogenous CD24 and FASL polypeptides, exogenous CD24 and CCL21 polypeptides, exogenous CD24 and Mfge8 polypeptides, and exogenous CD24 and Serpinb95 polypeptides, etc.

[0139] In some embodiments, the cell comprises a genome modification of one or more targeting polynucleotide sequences that regulate the expression of MHC I and / or MHC II. In some aspects, a gene editing system is used to modify one or more targeting polynucleotide sequences. In some embodiments, the targeting polynucleotide sequences are one or more selected from the group consisting of B2M, CIITA, and NLRC5. In certain embodiments, the genome of the cell is modified to reduce or delete key components of HLA expression.

[0140] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome that has been gene-edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. In certain aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome that has been gene-edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cells or populations thereof. In certain aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome in which one or more genes have been edited to delete contiguous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cells or populations thereof. In certain aspects, the present disclosure provides stem cells (e.g., pluripotent stem cells or induced pluripotent stem cells) or populations thereof comprising a genome in which one or more genes have been edited to delete contiguous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cells or populations thereof.

[0141] In certain embodiments, expression of MHC I or MHC II is modulated by targeting and deleting adjacent stretches of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M, CIITA, and NLRC5.

[0142] In some embodiments, the cells and methods described herein involve genome editing a human cell to cleave the CIITA gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M and NLRC5. In some embodiments, the cells and methods described herein involve genome editing a human cell to cleave the B2M gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, CIITA and NLRC5. In some embodiments, the cells and methods described herein involve genome editing a human cell to cleave the NLRC5 gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M and CIITA.

[0143] In certain embodiments, MHC I expression is regulated by overexpressing or increasing expression of DUX4. In some embodiments, the polynucleotide sequence encoding DUX4 is a codon-altered sequence that includes one or more base substitutions to reduce the total number of CpG sites while maintaining the DUX4 protein sequence. In some cases, the codon-altered sequence is SEQ ID NO: 1. In other cases, the polynucleotide sequence encoding DUX4 is a nucleotide sequence that encodes a polypeptide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-25. In some embodiments, the cells described herein that have increased expression of DUX4 also overexpress CD24.

[0144] In some embodiments, the cells described herein include, but are not limited to, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and their progeny. In some embodiments, the present disclosure provides stem cells (e.g., hypoimmunogenic stem cells, pluripotent stem cells, adult stem cells, and hematopoietic stem cells) or populations thereof that have been modified as described herein.

[0145] In some embodiments, the primary T cells are selected from the group comprising cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor infiltrating lymphocytes, and combinations thereof.

[0146] In some embodiments, the primary T cells are from a pool of primary T cells from one or more donor subjects different from the recipient subject (e.g., the patient receiving the cells). Primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects and pooled together. In some embodiments, the primary T cells are harvested from one or more individuals, and in some instances, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells or pool of primary T cells are engineered to exogenously express CD24, and optionally, CD47, and cultured in vitro.

[0147] In certain embodiments, primary T cells or a pool of primary T cells are engineered to express a chimeric antigen receptor (CAR). The CAR can be any known to those skilled in the art. Useful CARs include those that bind to antigens selected from the group consisting of CD19, CD38, CD123, CD138, and BCMA. In some cases, the CAR is the same as or equivalent to those used in FDA-approved CAR-T cell therapies, such as tisagenlecleucel and axicabtagene ciloleucel, or others currently being investigated in clinical trials.

[0148] In some embodiments, the primary T cells or pool of primary T cells are engineered to exhibit reduced expression of an endogenous T cell receptor compared to unmodified primary T cells. In certain embodiments, the primary T cells or pool of primary T cells are engineered to exhibit reduced expression of CTLA4, PD1, or both CTLA4 and PD1 compared to unmodified primary T cells. Methods for genetically modifying cells, including T cells, are described in detail, for example, in WO2016 / 183041, the disclosure of which is incorporated herein by reference in its entirety, including tables, appendices, sequence listings, and figures.

[0149] In some embodiments, the CAR T cells comprise a CAR selected from the group comprising: (a) a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces cytokine gene expression upon successful signaling of the CAR.

[0150] In some embodiments, the antigen binding domain of the CAR is selected from the group including, but not limited to, (a) an antigen binding domain that targets an antigen characteristic of a neoplastic cell, (b) an antigen binding domain that targets an antigen characteristic of a T cell, (c) an antigen binding domain that targets an antigen characteristic of an autoimmune or inflammatory disease, (d) an antigen binding domain that targets an antigen characteristic of a senescent cell, (e) an antigen binding domain that targets an antigen characteristic of an infectious disease, and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

[0151] In some embodiments, the antigen-binding domain is selected from the group including an antibody, an antigen-binding portion thereof, an scFv, and an Fab. In some embodiments, the antigen-binding domain binds to CD19 or BCMA. In some embodiments, the antigen-binding domain is an anti-CD19 scFv, such as, but not limited to, FMC63.

[0152] In some embodiments, the transmembrane domain of the CAR comprises one selected from the group comprising TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0153] In some embodiments, the signaling domain of the CAR comprises a costimulatory domain. For example, the signaling domain can include a costimulatory domain. Alternatively, the signaling domain can include one or more costimulatory domains. In certain In embodiments, the signaling domain comprises a costimulatory domain. In other embodiments, the signaling domain comprises a costimulatory domain. In some cases, when a CAR comprises two or more costimulatory domains, the two costimulatory domains are not the same. In some embodiments, the costimulatory domain comprises two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation. Enhances T cell persistence.

[0154] As described herein, a fourth-generation CAR can contain an antigen-binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces cytokine gene expression upon successful CAR signaling. In some cases, the cytokine gene is an endogenous or exogenous cytokine gene of the low-immunogenic cell. In some cases, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is selected from the group including IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces cytokine gene expression upon successful CAR signaling comprises a transcription factor or a functional domain or fragment thereof.

[0155] In some embodiments, the CAR comprises a CD3 zeta domain, or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In certain embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene. In some embodiments, the CAR comprises (i) an anti-CD19 scFv, (ii) a CD8α hinge and transmembrane domain, or a functional variant thereof, (iii) a 4-1BB costimulatory domain, or a functional variant thereof, and (iv) a CD3ζ signaling domain, or a functional variant thereof.

[0156] Methods for introducing CAR constructs or generating CAR-T cells are well known to those skilled in the art.Detailed descriptions can be found, for example, in Vormittag et al., Curr Opin Biotechnol, 2018, 53, 162-181 and Eyquem et al., Nature, 2017, 543, 113-117.

[0157] In some embodiments, cells derived from primary T cells comprise reduced expression of an endogenous T cell receptor, e.g., by disruption of the endogenous T cell receptor gene (e.g., T cell receptor alpha constant region (TRAC) or T cell receptor beta constant region (TRBC)). In some embodiments, an exogenous nucleic acid encoding a polypeptide disclosed herein (e.g., a chimeric antigen receptor, CD24, CD47, or another tolerogenic factor disclosed herein) is inserted into the disrupted T cell receptor gene.

[0158] In some embodiments, cells derived from primary T cells comprise reduced expression of cytotoxic T lymphocyte-associated protein 4 (CTLA4) and / or programmed cell death (PD1). Methods for reducing or eliminating the expression of CTLA4, PD1, or both CTLA4 and PD1 can include any of the art-recognized methods, such as, but not limited to, genetic modification techniques using rare-cutting endonucleases and RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nuclease, meganuclease, and homing endonucleases.

[0159] In some embodiments, the populations of engineered cells described induce reduced levels of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the cells induce reduced levels of systemic TH1 activation or no systemic TH1 activation in the recipient subject. In some embodiments, the cells induce reduced levels of immune activation of peripheral blood mononuclear cells (PBMCs) or no immune activation of PBMCs in the recipient subject. In some embodiments, the cells induce reduced levels of donor-specific IgG antibodies against the cells or no donor-specific IgG antibodies upon administration to the recipient subject. In some embodiments, the cells induce reduced levels of IgM and IgG antibody production against the cells or no IgM and IgG antibody production in the recipient subject. In some embodiments, the cells induce reduced levels of cytotoxic T cell killing of the cells upon administration to a recipient subject.

[0160] B.CD24 In some aspects, the present disclosure provides stem cells (e.g., pluripotent stem cells or induced pluripotent stem cells) or populations thereof that have been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide) CD24. In some aspects, the present disclosure provides methods for modifying the stem cell genome to express CD24. In some embodiments, the stem cells express exogenous CD24. In some cases, the stem cells express an expression vector comprising a nucleotide sequence encoding a human CD24 polypeptide.

[0161] CD24, also known as the heat-stable antigen or small cell lung cancer cluster 4 antigen, is a glycosylated glycosylphosphatidylinositol-anchored surface protein (Pirruccello et al., J Immunol, 1986, 136, 3779-3784; Chen et al., Glycobiology, 2017, 57, 800-806). It binds to Siglec-10 on innate immune cells. Recently, CD24, via Siglec-10, has been shown to function as an innate immune checkpoint (Barkal et al., Nature, 2019, 572, 392-396).

[0162] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the cells comprise a nucleotide sequence encoding a CD24 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the cells comprise a nucleotide sequence encoding a CD24 polypeptide having a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0163] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1. In some cases, the CD24 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1.

[0164] In some embodiments, the cells comprise a nucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3. In some embodiments, the cells comprise a nucleotide sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3.

[0165] In another embodiment, CD24 protein expression is detected using Western blots of cell lysates probed with an antibody against CD24 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD24 mRNA.

[0166] C.CIITA In certain embodiments, the inventions disclosed herein regulate (e.g., reduce or eliminate) expression of MHC II genes by targeting and regulating (e.g., reducing or eliminating) class II transactivator (CIITA) expression. In some embodiments, regulation 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, which regulates MHC II transcription by binding to the MHC enhanceosome.

[0167] In some embodiments, the target polynucleotide sequence of the present invention is a variant of CIITA. In some embodiments, the target polynucleotide sequence is a homolog of CIITA. In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

[0168] In some embodiments, reducing or eliminating the expression of CIITA reduces or eliminates the expression of one or more of the following MHC class II: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0169] In some embodiments, the cells described herein comprise a genetic modification that targets the CIITA gene. In some embodiments, the genetic modification that targets the CIITA gene with a rare-cutting endonuclease comprises a Cas protein or a Cas protein. The present invention relates to a method for the preparation of a CIITA gene comprising the steps of: (a) preparing a CIITA gene; (b) preparing a CIITA gene; and (c) preparing a CIITA gene; and (d) preparing a CIITA gene; and (e) preparing a CIITA gene; and (f) preparing a CIITA gene; and (g) preparing a CIITA gene; and (h) preparing a CIITA gene; and (i) preparing a CIITA gene; and (ii) preparing a CIITA gene; and (iii) preparing a CIITA gene; and (iv) preparing a CIITA gene; and (v) preparing a CIITA gene; and (vi ...

[0170] The assay for testing whether CIITA gene is inactivated is known and described herein.In one embodiment, the resulting genetic modification of CIITA gene and the reduction of HLA-II expression can be assayed by FACS analysis by PCR.In another embodiment, CIITA protein expression is detected by Western blot of cell lysate probed with antibody against CIITA protein.In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modification.

[0171] D.B2M In certain aspects, the inventions disclosed herein modulate (e.g., reduce or eliminate) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of the accessory chain B2M. In some aspects, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) the expression of B2M, surface trafficking of MHC-I molecules is blocked, and such cells exhibit immune tolerance when transplanted into a recipient subject. In some embodiments, the cells are considered, for example, to be hypoimmunogenic in the recipient subject or patient upon administration.

[0172] In some embodiments, the target polynucleotide sequence of the present invention 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.

[0173] In some embodiments, reducing or eliminating the expression of B2M reduces or eliminates the expression of one or more of the following MHC I molecules - HLA-A, HLA-B, and HLA-C.

[0174] In some embodiments, the hypoimmunogenic cells described herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, 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 to 85644 in Appendix 2 or Table 15 of WO2016 / 183041, the disclosure of which is incorporated herein by reference in its entirety.

[0175] Assays for testing whether the B2M gene is inactivated are known and described herein. In one embodiment, the resulting genetic modification of the B2M gene and the reduction of HLA-I expression can be assayed by FACS analysis by PCR. In another embodiment, B2M protein expression is detected using Western blot of cell lysate probed with an antibody against B2M protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0176] E.NLRC5 In certain embodiments, the invention disclosed herein targets and modulates (e.g., inhibits) the expression of the NLR family, CARD domain-containing 5 / NOD27 / CLR16.1 (NLRC5). In some embodiments, the expression of MHC-I genes is regulated (e.g., reduced or eliminated) by regulating (e.g., reducing or eliminating). In some embodiments, the regulation occurs using a CRISPR / Cas system. NLRC5 is a key regulator of MHC-I-mediated immune responses, and like CIITA, NLRC5 is highly induced by IFN-γ and can translocate to the nucleus. NLRC5 activates the promoter of MHC-I genes and induces the transcription of MHC-I and related genes involved in MHC-I antigen presentation.

[0177] In some embodiments, the target polynucleotide sequence of the present invention 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.

[0178] In some embodiments, reducing or eliminating the expression of NLRC5 reduces or eliminates the expression of one or more of the following MHC I molecules - HLA-A, HLA-B, and HLA-C.

[0179] In some embodiments, the cells described herein comprise a genetic modification targeting the NLRC5 gene. In some embodiments, the genetic modification targeting the NLRC5 gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene. In some embodiments, 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 to 81239 in Appendix 3 or Table 14 of WO2016 / 183041, the disclosure of which is incorporated herein by reference in its entirety.

[0180] Assays for testing whether the NLRC5 gene is inactivated are known and described herein.In one embodiment, the resulting genetic modification of the NLRC5 gene and the reduction in HLA-I expression can be assayed by FACS analysis using PCR.In another embodiment, NLRC5 protein expression is detected using Western blot of cell lysates probed with an antibody against NLRC5 protein.In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modification.

[0181] F.CD47 In some embodiments, the cells comprise an exogenous CD24 polypeptide and an exogenous CD47 polypeptide. In some embodiments, the pluripotent cells or differentiated cells generated from the pluripotent cells comprise an exogenous CD24 polypeptide and an exogenous CD47 polypeptide.

[0182] In some aspects, the present disclosure provides cells or populations thereof that have been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide) CD47. In some aspects, the present disclosure provides methods for modifying a cell genome to express CD47. In some embodiments, the stem cells express exogenous CD47 polynucleotides and / or polypeptides. In some cases, the cells express an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide.

[0183] CD47 is a leukocyte surface antigen that plays a role in cell adhesion and integrin regulation. It is expressed on the surface of cells and signals circulating cells not to ingest the cells.

[0184] In some embodiments, the cells outlined herein are identified under NCBI reference number NP_00 1768.1 and NP_942088.1 and SEQ ID NOs: 32 and 33. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1 and SEQ ID NOs: 32 and 33. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1 and SEQ ID NOs: 32 and 33. In some embodiments, the cells comprise a nucleotide sequence of 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 reference numbers NM_001777.3 and NM_198793.2. In some embodiments, the cells comprise a nucleotide sequence of CD47 set forth in NCBI reference numbers NM_001777.3 and NM_198793.2.

[0185] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1 and SEQ ID NOs: 32 and 33. In some embodiments, the cells outlined herein comprise a CD47 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1 and SEQ ID NOs: 32 and 33.

[0186] In some embodiments, a gene editing system, such as a CRISPR / Cas system, is used to facilitate insertion of a tolerogenic factor, such as insertion of a tolerogenic factor into a safe harbor locus, such as the AAVS1 locus. In some cases, a polynucleotide sequence encoding CD47 is inserted into a safe harbor locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, or SHS231 locus.

[0187] In another embodiment, CD47 protein expression is detected using Western blots of cell lysates probed with an antibody against CD47 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD47 mRNA.

[0188] G.DUX4 In some embodiments, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome modified to increase expression of a tolerogenic or immunosuppressive factor gene, such as DUX4. In some embodiments, the present disclosure provides methods for modifying a cell's genome to provide increased expression of DUX4. In one embodiment, the present disclosure provides cells or populations thereof comprising exogenously expressed DUX4 protein. In some embodiments, increased expression of DUX4 reduces or eliminates expression of one or more of the following MHC I molecules—HLA-A, HLA-B, and HLA-C.

[0189] In some embodiments, the cells comprise an exogenous CD24 polypeptide and an exogenous DUX4 polypeptide. In some embodiments, the pluripotent cells or differentiated cells generated from the pluripotent cells comprise an exogenous CD24 polypeptide and an exogenous DUX4 polypeptide.

[0190] DUX4 is active in embryonic tissues and induced pluripotent stem cells and is suppressed in normal, healthy somatic cells. DUX4 is a transcription factor that silences IFN-gamma-mediated induction of major histocompatibility complex (MHC) class I gene expression (e.g., B2M, HLA-A, HLA-B, and HLA-C expression) (Feng et al., 2015, ELife4; De Iaco et al., 2017, Nat Genet, 49, 941-945; Hendrickson et al., 2017, Nat Genet, 49, 925-934; Snider et al., 2010, PLoS Genet, e1001181; Whiddon et al., 2017, Nat Genet). DUX4 expression acts to block IFN-gamma-mediated induction of major histocompatibility complex (MHC) class I gene expression (e.g., B2M, HLA-A, HLA-B, and HLA-C expression). DUX4 expression is associated with suppressed antigen presentation by MHC class I (Chew et al., Developmental Cell, 2019, 50, 1-14). DUX4 functions as a transcription factor in the cleavage stage gene expression (transcription) program, and its target genes include, but are not limited to, coding genes, non-coding genes, and repetitive elements.

[0191] DUX4 has at least two isoforms, the longest of which comprises the C-terminal transcriptional activation domain of DUX4. Isoforms are produced by alternative splicing. See, e.g., Geng et al., 2012, Dev Cell, 22, 38-51; Snider et al., 2010, PLoS Genet, e1001181. The active isoform of DUX4 contains the N-terminal DNA-binding domain and the C-terminal activation domain. See, e.g., Choi et al., 2016 Nucleic Acid Res, 44, 5161-5173.

[0192] It has been shown that reducing the number of CpG motifs in DUX4 reduces silencing of the DUX4 transgene (Jagannathan et al., Human Molecular Genetics, 2016, 25(20):4419-4431). SEQ ID NO: 1 represents a codon-altered sequence of DUX4 containing one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. The nucleic acid sequence is commercially available from Addgene under catalog number 99281.

[0193] In certain embodiments, at least one or more polynucleotides can be utilized to facilitate insertion of DUX4 into a cell, such as a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell.

[0194] In some embodiments, a gene editing system, such as a CRISPR / Cas system, is used to facilitate insertion of a tolerogenic factor, such as insertion of a tolerogenic factor into a safe harbor locus, such as the AAVS1 locus. In some cases, a polynucleotide sequence encoding DUX4 is inserted into a safe harbor locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, or SHS231 locus.

[0195] In some instances, the polynucleotide sequence encoding DUX4 is SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. In some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

[0196] In other embodiments, expression of the tolerogenic factor is promoted using an expression vector. In some embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, which is a codon-altered sequence comprising one or more base substitutions to reduce the total number of CpG sites while maintaining the DUX4 protein sequence. In some cases, the codon-altered sequence of DUX4 is SEQ ID NO: 1. In other embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, which is SEQ ID NO: 1. In some embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, which is a nucleotide sequence encoding a polypeptide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. In some embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, which is a nucleotide sequence encoding a polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

[0197] Increased DUX4 expression can be assayed using known techniques such as Western blot, ELISA assay, FACS assay, immunoassay, and the like.

[0198] H. Additional Tolerogenic Factors In certain embodiments, one or more tolerogenic factors can be inserted or reinserted into genome-edited cells to create immune-privileged universal donor cells, such as universal donor stem cells, universal donor T cells, or universal donor cells. In certain embodiments, the hypoimmunogenic cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of CD24, CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9. In some embodiments, the tolerogenic factor is selected from the group consisting of CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9.

[0199] In some embodiments, a gene editing system, such as a CRISPR / Cas system, is used to facilitate the insertion of a tolerogenic factor, such as the insertion of a tolerogenic factor into a safe harbor locus, such as the AAVS1 locus. In some cases, the polynucleotide sequence encoding any of the tolerogenic factors described herein can be, but is not limited to, However, they are inserted into safe harbor loci such as the AAVS1, CCR5, CLYBL, ROSA26, or SHS231 loci.

[0200] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome modified to express CD47. In some aspects, the present disclosure provides methods for modifying a cell genome to express CD47. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of CD47 into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 200784-231885 in Table 29 of WO2016 / 183041, incorporated herein by reference.

[0201] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome in which the cellular genome has been modified to express HLA-C. In some aspects, the present disclosure provides methods for modifying a cellular genome to express HLA-C. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of HLA-C into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3278-5183 in Table 10 of WO2016 / 183041, which is incorporated herein by reference.

[0202] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome in which the cellular genome has been modified to express HLA-E. In some aspects, the present disclosure provides methods for modifying a cellular genome to express HLA-E. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of HLA-E into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 189859-193183 in Table 19 of WO2016 / 183041, which is incorporated herein by reference.

[0203] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome modified to express HLA-F. In some aspects, the present disclosure provides methods for modifying a cell genome to express HLA-F. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of HLA-F into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 688808-399754 in Table 45 of WO2016 / 183041, incorporated herein by reference.

[0204] In some aspects, the present disclosure provides a cell (e.g., a stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a differentiated cell derived from or produced from such a stem cell, a hematopoietic stem cell, a primary T cell, a chimeric antigen receptor agonist ... In some embodiments, the present disclosure provides methods for modifying a cell genome to express HLA-G. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of HLA-G into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 188372-189858 in Table 18 of WO2016 / 183041, which is incorporated herein by reference.

[0205] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome that has been modified to express PD-L1. In some aspects, the present disclosure provides methods for modifying a cell's genome to express PD-L1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of PD-L1 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 193184-200783 in Table 21 of WO2016 / 183041, which is incorporated herein by reference.

[0206] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome in which the cellular genome has been modified to express CTLA4-Ig. In some aspects, the present disclosure provides methods for modifying a cellular genome to express CTLA4-Ig. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of CTLA4-Ig into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any disclosed in WO2016 / 183041, including the sequence listing.

[0207] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome modified to express a CI-inhibitor. In some aspects, the present disclosure provides methods for modifying a cell genome to express a CI-inhibitor. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate the insertion of a CI-inhibitor into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any of those disclosed in WO2016 / 183041, including the sequence listing.

[0208] In some aspects, the present disclosure provides cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof comprising a genome in which the cellular genome has been modified to express IL-35. In some aspects, the present disclosure provides methods for modifying a cellular genome to express IL-35. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of IL-35 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one disclosed in WO2016 / 183041, including the sequence listing.

[0209] In some embodiments, the tolerogenic factor is expressed in cells using an expression vector. For example, an expression vector for expressing CD47 in cells comprises a polynucleotide sequence encoding a CD47 polypeptide. In some embodiments, the CD47 polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. The expression vector can be an inducible expression vector. The expression vector can be a viral vector, such as, but not limited to, a lentiviral vector.

[0210] In some embodiments, the present disclosure provides that the cellular genome is selected from the group consisting of HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, L Provided are cells (e.g., stem cells, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced by such stem cells, hematopoietic stem cells, primary T cells, chimeric antigen receptor (CAR) T cells, and any progeny thereof) or populations thereof, comprising a genome that has been modified to express any one of the polypeptides selected from the group consisting of FA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, and IDO1. In some aspects, the present disclosure provides methods for modifying a cell genome to express any one of polypeptides selected from the group consisting of HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, and IDO1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be utilized to facilitate insertion of a selected polypeptide into the stem cell line, hi certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one of those disclosed in Appendices 1-47 and the Sequence Listing of WO2016 / 183041, the disclosures of which are incorporated herein by reference.

[0211] I. Exemplary Embodiments In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules of the MHC class I complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules of the MHC class II complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules of MHC class II and the MHC class II complex.

[0212] In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of B2M. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules selected from B2M and CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules selected from B2M and NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules selected from CIITA and NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and decreased expression of one or more molecules selected from CIITA and NLRC5. and decreased expression of one or more molecules of B2M, CIITA, and NLRC5. Any of the cells described herein can also exhibit increased expression of one or more factors selected from the group consisting of, but not limited to, CD47, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9.

[0213] In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more molecules of the MHC class I complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more molecules of the MHC class II complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more molecules of the MHC class II and MHC class II complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of B2M. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more molecules of B2M and CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more of the molecules B2M and NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more of the molecules CIITA and NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD24 and CD47 and decreased expression of one or more of the molecules B2M, CIITA, and NLRC5. Any of the cells described herein may also exhibit increased expression of one or more selected from the group consisting of, but not limited to, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9.

[0214] Those skilled in the art will understand that expression levels, such as increased or decreased expression of an engineered or modified gene, protein, or molecule, can be referenced or compared to equivalent unengineered or unmodified cells. In some embodiments, engineered stem cells with increased expression of CD24 refer to modified stem cells that have higher levels of CD24 protein compared to unmodified stem cells.

[0215] J. Genetic Modification Methods In some embodiments, the rare-cutting endonuclease is introduced into cells containing a target polynucleotide sequence in the form of a nucleic acid encoding the rare-cutting endonuclease. The process of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA (e.g., synthetic modified mRNA) as described herein.

[0216] The present invention provides any method that can be used by a person skilled in the art using the CRISPR / Cas system of the present invention. It is contemplated that the target polynucleotide sequence is altered in the following manner. Any CRISPR / Cas system capable of altering a target polynucleotide sequence in a cell can be used. Such a CRISPR / Cas system can use a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;1(6)e60). The molecular machinery of such Cas proteins that enables the CRISPR / Cas system to alter a target polynucleotide sequence in a cell includes RNA-binding proteins, endonucleases and exonucleases, helicases, and polymerases. In some embodiments, the CRISPR / Cas system is a CRISPR type I system. In some embodiments, the CRISPR / Cas system is a CRISPR type II system. In some embodiments, the CRISPR / Cas system is a CRISPR type RV system.

[0217] The CRISPR / Cas system of the present invention can be used to modify any target polynucleotide sequence within a cell. Those skilled in the art will readily understand that the desired target polynucleotide sequence to be modified in any particular cell can correspond to any genomic sequence whose expression is associated with a disorder or otherwise facilitates pathogen entry into the cell. For example, the target polynucleotide sequence desired to be altered in the cell can be a polynucleotide sequence corresponding to a genomic sequence containing a disease-associated single nucleotide polymorphism. In such an example, the CRISPR / Cas system of the present invention can be used to correct the disease-associated SNP in the cell by replacing it with a wild-type allele. As another example, the polynucleotide sequence of a target gene involved in the entry or proliferation of a pathogen into a cell can be a suitable target for deletion or insertion to disrupt the function of the target gene to prevent the pathogen from entering the cell and replicating within it.

[0218] In some embodiments, 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.

[0219] In some embodiments, the CRISPR / Cas system of the present invention comprises a Cas protein and at least one to two ribonucleic acids capable of hybridizing the Cas protein to a target motif in a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) linked by peptide bonds, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, and analogs of the above.

[0220] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitutions and / or modifications can prevent or reduce proteolysis and / or extend the half-life of the polypeptide within a cell. In some embodiments, the Cas protein can comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein can comprise naturally occurring amino acids. In some embodiments, the Cas protein can comprise alternative amino acids (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein can comprise modifications to include moieties (e.g., pegylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0221] In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein comprises a Cas protein of the E. coli subtype (also known as CASS2). Exemplary Cas proteins of the E. coli subtype include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the 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, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the 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, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the 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, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the 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, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a.In some embodiments, the 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, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, 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).

[0222] In some embodiments, the Cas protein comprises any one of the Cas proteins described herein, or a functional portion thereof. As used herein, "functional portion" refers to a portion of a peptide that retains its ability to form a complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, 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. In some embodiments, the functional portion comprises a combination of operably linked Cas12a (also known as Cpf1) 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. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of a Cas12a protein comprises a functional portion of a RuvC-like domain.

[0223] In some embodiments, exogenous Cas proteins can be introduced into cells in the form of polypeptides. In certain embodiments, Cas proteins can be conjugated or fused to cell-penetrating polypeptides or cell-penetrating peptides. As used herein, "cell-penetrating polypeptides" and "cell-penetrating peptides" refer to polypeptides or peptides, respectively, that facilitate the uptake of molecules into cells. Cell-penetrating polypeptides can contain detectable labels.

[0224] In certain embodiments, the Cas protein can be conjugated or fused to a charged protein (e.g., having a positive charge, a negative charge, or an overall neutral charge). Such a linkage can be covalent. In some embodiments, the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al. ACS Chem Biol. 2010;5(8):747-52). In certain embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include Tat, oligoarginines, and penetratin. In some embodiments, 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 penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a positively supercharged GFP. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a penetratin domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a positively supercharged GFP.

[0225] In some embodiments, the Cas protein is introduced into a cell containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The process of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA (e.g., synthetic modified mRNA) as described herein.

[0226] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. 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 (e.g., a synthetic, modified mRNA) as described herein.

[0227] The methods of the present invention contemplate the use of any ribonucleic acid that can direct a Cas protein to and hybridize with a target motif in a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises a tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that directs a Cas protein to and hybridizes with a target motif in a target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs a Cas protein to and hybridizes with a target motif in a target polynucleotide sequence in a cell. In some embodiments, both of the one or two ribonucleic acids comprise a guide RNA that directs a Cas protein to and hybridizes with a target motif in a target polynucleotide sequence in a cell. The ribonucleic acids of the present invention can be selected to hybridize with a variety of different target motifs, depending on the particular CRISPR / Cas system used and the sequence of the target polynucleotide, as will be appreciated by those of skill 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. In some embodiments, the one to two ribonucleic acids hybridize to a target motif containing at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif containing at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein adjacent to a mutant allele located between the target motifs.

[0228] In some embodiments, each of the one to two ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif in a target polynucleotide sequence in a cell.

[0229] In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize with sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize with sequences on opposite strands of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are not complementary to and / or hybridize with sequences on opposite strands of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize with overlapping target motifs of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize with offset target motifs of a target polynucleotide sequence.

[0230] In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one to two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. 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 (e.g., a synthetic, modified mRNA) as described herein.

[0231] Exemplary gRNA sequences useful for CRISPR / Cas-based targeting of the genes described herein are provided in Table 1. The sequences can be found in WO2016 / 183041, filed May 9, 2016, the disclosure of which, including tables, appendices, and sequence listings, is incorporated herein by reference in its entirety. [Table 1]

[0232] In some embodiments, the cells of the invention are generated using transcription activator-like effector nuclease (TALEN) methodology.

[0233] The term "TALE-nuclease" (TALEN) refers to a fusion protein consisting of a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and a single nuclease catalytic domain for cleaving a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain, more preferably a domain with endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain can be fused to a meganuclease, such as I-CreI and I-OnuI, or functional variants thereof. In a more preferred embodiment, the 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 fusion of the catalytic domain of I-TevI ​​with engineered TAL repeats, as described in WO2012 / 138927. Transcription activator-like effectors (TALEs) are proteins from the bacterial species Xanthomonas that contain multiple repeats, each containing two residues (RVDs) at positions 12 and 13 that are specific for each nucleotide base of a nucleic acid targeting sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBDs) can also be derived from novel modular proteins recently discovered by the applicant in different bacterial species. A modular protein has the advantage of exhibiting more sequence variability than TAL repeats. Preferably, the RVDs involved in the recognition of 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. In another embodiment, the critical amino acids 12 and 13 can be mutated to other amino acid residues to adjust their specificity for the nucleotides A, T, C, and G, and in particular to enhance this specificity. TALEN kits are commercially available.

[0234] In some embodiments, cells are engineered using zinc finger nucleases (ZFNs). A "zinc finger binding protein" is a protein or polypeptide that binds to DNA, RNA, and / or proteins, preferably in a sequence-specific manner, as a result of stabilization of the protein structure by the coordination of zinc ions. The term zinc finger binding protein is often abbreviated as zinc finger protein or ZFP. Individual DNA-binding domains are typically referred to as "finger(s)." ZFPs have at least one finger, typically two, three, or six fingers. Each finger binds to two to four base pairs of DNA, typically three to four base pairs. ZFPs bind to nucleic acid sequences called target sites or target segments. Each finger typically consists of approximately 30 amino acids, a zinc-chelating, DNA-binding subdomain. Studies have shown that this class of single zinc finger consists of an alpha helix containing two invariant histidine residues that coordinate zinc with two cysteine ​​residues in a single beta turn (see, e.g., Berg & Shi, Science 271:1081-1085 (1996)).

[0235] In some embodiments, the cells of the present invention are generated using a homing endonuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and generate single- or double-strand breaks. Homing endonucleases are highly specific and recognize DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. Homing endonucleases according to the present invention can correspond to, for example, LAGLIDADG endonuclease, HNH endonuclease, or GIY-YIG endonuclease. A preferred homing endonuclease according to the present invention can be an I-CreI variant.

[0236] In some embodiments, the cells of the invention are produced using meganucleases, which by definition are sequence-specific endonucleases that recognize large sequences (Chevalier, B.S. and B.L. Stoddard, Nucleic Acids, vol. 14, no. 1, pp. 111-114, 2002). Acids Res., 2001, 29, 3757-3774). They can cleave unique sites in living cells, thereby enhancing gene targeting by more than 1000-fold near the cleavage site (Puchta et al., Nucleic Acids Res., 2001, 29, 3757-3774). Res.,1993,21,5034-5040, Rouet et al.,Mol.Cell.Biol.,1994,14,8096-8106, Choulika et al.,Mol.Cell.Biol.,1995,15,1968-1973, Puchta et al. al.,Proc.Natl.Acad.Sci.USA,1996,93,5055-5060,Sargent et al.,Mol.Cell.Biol.,1997,17,267-77,Donoho et al.,Mol.Cell.Biol,1998,18,4070-4078,Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101, Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448).

[0237] In some embodiments, the cells of the present invention are generated using RNA silencing or RNA interference (RNAi) to knock down (e.g., reduce, eliminate, or inhibit) the expression of a polypeptide, such as an immune tolerogenic factor. Useful RNAi methods include those utilizing synthetic RNAi molecules, short interfering RNA (siRNA), PIWI-interacting RNA (piRNA), short hairpin RNA (shRNA), microRNA (miRNA), and other art-recognized transient knockdown methods. RNAi reagents, including sequence-specific shRNA, siRNA, miRNA, etc., are commercially available. For example, CIITA can be knocked down in pluripotent stem cells by introducing CIITA siRNA or a CIITA shRNA-expressing virus into cells. In some embodiments, RNA interference is used to reduce or inhibit the expression of at least one selected from the group consisting of CIITA, B2M, and NLRC5.

[0238] In some embodiments, the cells of the present invention are genetically modified to reduce the expression of one or more immune factors (including target polypeptides), thereby creating immune-privileged or hypoimmunogenic cells.In certain embodiments, the cells disclosed herein (such as stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells and CAR-T cells) comprise one or more genetic modifications to reduce the expression of one or more target polynucleotides.Non-limiting examples of such target polynucleotides and polypeptides include CIITA, B2M, NLRC5, CTLA4, PD1, HLA-A, HLA-BM, HLA-C, RFX-ANK, NFY-A, RFX5, RFX-AP, NFY-B, NFY-C, IRF1 and TAP1.

[0239] In some aspects, gene modification occurs using CRISPR / Cas system.By regulating (for example, reducing or deleting) the expression of one or more target polynucleotides, such cells exhibit reduced immune activation when transplanted into recipient subject.In some embodiments, the cells are considered, for example, to be low immunogenic in recipient subject or patient when administered.

[0240] K. Methods for overexpression of tolerogenic factors Provided herein are cells that do not induce or activate an immune response upon administration to a recipient subject. As described above, in some embodiments, the cells are modified to increase the expression of genes and tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in the recipient.

[0241] In certain embodiments, any of the cells disclosed herein (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells, and CAR-T cells) having a genetic modification that regulates the expression of one or more target proteins listed herein are also modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of CD24, CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4, C1-inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, and Serpinb9. In some embodiments, the tolerogenic factor is CD24, CD47, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4, C1-inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, and Serpinb9 is selected from the group including:

[0242] Useful genome, polypeptide, and polypeptide information for human CD27 (also known as CD27L receptor, tumor necrosis factor receptor superfamily member 7, TNFSF7, T-cell activation antigen S152, Tp55, and T14) is provided, for example, at GeneCard identifier GC12P008144, HGNC No. 11922, NCBI Gene ID 939, Uniprot No. P26842, and NCBI RefSeq Nos. NM_001242.4 and NP_001233.1.

[0243] Useful genome, polynucleotide, and polypeptide information for human CD46 is available, for example, under GeneCard identifier GC01P207752, HGNC No. 6953, NCBI Gene ID 4179, Uniprot No. P15529, and NCBI RefSeq Nos. NM_002389.4, NM_153826.3, NM_172350.2, NM_172351.2, NM_172352.2. Available in NP_758860.1, NM_172353.2, NM_172359.2, NM_172361.2, 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.

[0244] Useful genomic, polynucleotide, and polypeptide information for human CD55 (also known as complement decay-accelerating factor) is provided, for example, at GeneCard identifier GC01P207321, HGNC No. 2665, NCBI Gene ID 1604, Uniprot No. P08174, and NCBI RefSeq Nos. NM_000574.4, NM_001114752.2, NM_001300903.1, NM_001300904.1, NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.

[0245] Useful genome, polynucleotide, and polypeptide information for human CD59 is available, for example, at GeneCard identifier GC11M033704, HGNC No. 1689, NCBI Gene ID 966, Uniprot No. P13987, and NCBI RefSeq Provided in Nos. NP_000602.1, NM_000611.5, NP_001120695.1, NM_001127223.1, NP_001120697.1, NM_001127225.1, NP_001120698.1, NM_001127226.1, NP_001120699.1, NM_001127227.1, NP_976074.1, NM_203329.2, NP_976075.1, NM_203330.2, NP_976076.1, and NM_203331.2.

[0246] Useful genomic, polynucleotide, and polypeptide information for human CD200 is provided, for example, at GeneCard identifier GC03P112332, HGNC No. 7203, NCBI Gene ID 4345, Uniprot No. P41217, and NCBI RefSeq Nos. NP_001004196.2, NM_001004196.3, NP_001305757.1, NM_001318828.1, NP_005935.4, NM_005944.6, XP_005247539.1, and XM_005247482.2.

[0247] Useful genome, polynucleotide, and polypeptide information for human HLA-C is available, for example, from GeneCard identifier GC06M031272, HGNC No. 4933, NCBI Gene ID 3107, Uniprot No. P10321. and NCBI RefSeq Nos. NP_002108.4 and NM_002117.5.

[0248] Useful genomic, polynucleotide, and polypeptide information for human HLA-E is provided, for example, under GeneCard identifier GC06P047281, HGNC No. 4962, NCBI Gene ID 3133, Uniprot No. P13747, and NCBI RefSeq Nos. NP_005507.3 and NM_005516.5.

[0249] Useful genomic, polynucleotide, and polypeptide information for human HLA-G is provided, for example, at GeneCard identifier GC06P047256, HGNC No. 4964, NCBI Gene ID 3135, Uniprot No. P17693, and NCBI RefSeq Nos. NP_002118.1 and NM_002127.5.

[0250] Useful genomic, polynucleotide, and polypeptide information regarding human PD-L1 or CD274 can be found, for example, under GeneCard identifier GC09P005450, HGNC No. 17635, NCBI Gene ID 29126, Uniprot No. Q9NZQ7, and NCBI RefSeq Nos. NP_001254635.1, NM_001267706.1, NP_054862.1, and NM_014143.3.

[0251] Useful genomic, polynucleotide, and polypeptide information for human IDO1 is provided, for example, at GeneCard identifier GC08P039891, HGNC No. 6059, NCBI Gene ID 3620, Uniprot No. P14902, and NCBI RefSeq Nos. NP_002155.1 and NM_002164.5.

[0252] Useful genomic, polynucleotide, and polypeptide information for human IL-10 is provided, for example, at GeneCard identifier GC01M206767, HGNC No. 5962, NCBI Gene ID 3586, Uniprot No. P22301, and NCBI RefSeq Nos. NP_000563.1 and NM_000572.2.

[0253] Useful genomic, polynucleotide, and polypeptide information for human Fas ligand (also known as FasL, FASLG, CD178, TNFSF6, etc.) is provided, for example, at 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.

[0254] Useful genome, polynucleotide, and polypeptide information for human CCL21 is provided, for example, at GeneCard identifier GC09M034709, HGNC No. 10620, NCBI Gene ID 6366, Uniprot No. O00585, and NCBI RefSeq Nos. NP_002980.1 and NM_002989.3.

[0255] Useful genome, polynucleotide, and polypeptide information for human CCL22 is available, for example, from GeneCard identifier GC16P057359, HGNC No. 10621, NCBI Gene ID 6367, Uniprot No. O00626. and NCBI RefSeq Nos. NP_002981.2, NM_002990.4, XP_016879020.1, and XM_017023531.1.

[0256] Useful genome, polynucleotide, and polypeptide information for human Mfge8 is provided, for example, at GeneCard identifier GC15M088898, HGNC No. 7036, NCBI Gene ID 4240, Uniprot No. Q08431, and NCBI RefSeq Nos. NP_001108086.1, NM_001114614.2, NP_001297248.1, NM_001310319.1, NP_001297249.1, NM_001310320.1, NP_001297250.1, NM_001310321.1, NP_005919.2, and NM_005928.3.

[0257] Useful genome, polynucleotide, and polypeptide information for human SerpinB9 is provided, for example, at GeneCard identifier GC06M002887, HGNC No. 8955, NCBI Gene ID 5272, Uniprot No. P50453, and NCBI RefSeq Nos. NP_004146.1, NM_004155.5, XP_005249241.1, and XM_005249184.4.

[0258] Useful genomic, polynucleotide, and polypeptide information for human CD35 (also known as complement receptor type 1 (CR1), C3b / C4b receptor (C3BR), C4Br, knops blood group antigen, and C3 binding domain) is provided, for example, at GeneCard identifier GCO1P207496, HGNC No. 2334, NCBI Gene ID 1378, Uniprot No. P17927, and NCBI RefSeq Nos. NP_000564.2, NM_000573.3, NP_000642.3, and NM_000651.4.

[0259] Methods for regulating the expression of genes and factors (proteins) include genome editing techniques, and RNA or protein expression techniques, etc. For all of these techniques, well-known recombinant techniques are used to generate recombinant nucleic acids as outlined herein.

[0260] In certain embodiments, a recombinant nucleic acid encoding a tolerogenic factor may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence will generally be appropriate for the host cell and recipient to be treated. Many types of suitable expression vectors and appropriate regulatory sequences are known in the art for various host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcriptional start and stop sequences, translational start and stop sequences, and an enhancer or activator sequence. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of two or more promoters. The expression construct may reside in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In certain embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Certain embodiments include expression vectors containing a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers, and other expression control elements. In certain embodiments, an expression vector is used to encode a gene for a host cell to be transformed, a particular gene for which expression is desired, and a gene for which expression is desired. The vector may be designed for selection of the expression of another protein encoded therein, such as a mutant polypeptide, vector copy number, the ability to control that copy number, or antibiotic markers.

[0261] Examples of suitable mammalian promoters include, for example, promoters from the following genes: hamster ubiquitin / S27a promoter (WO 97 / 15664), simian vacuolar virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus (RSV) long terminal repeat region, mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus long terminal repeat region, and human cytomegalovirus (CMV) early promoter. Other heterologous mammalian promoters include actin, immunoglobulin, or heat shock promoters. In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyomavirus, fowlpox virus (UK 2,211,504 published July 5, 1989), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). In a further embodiment, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters, and heat shock promoters. The early and late promoters of SV40 can be conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral replication origin (Fiers et al., Nature 273:113-120 (1978)). The immediate early promoter of human cytomegalovirus can be conveniently obtained as a Hindlll E restriction fragment (Greenaway et al., Gene 18:355-360 (1982)). The above-mentioned references are incorporated by reference in their entirety.

[0262] In some embodiments, expression of a target gene (e.g., CD24, CD47, or another tolerogenic factor) is increased by expression of a fusion protein or protein complex containing (1) a site-specific binding domain specific for endogenous CD24, CD47, or other gene, and (2) a transcriptional activator.

[0263] In some embodiments, the regulatory element is comprised of a site-specific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA). In some embodiments, this method is achieved by a site-specific DNA-binding targeting protein, such as a zinc finger protein (ZFP), also known as a zinc finger nuclease (ZFN), or a fusion protein containing a ZFP.

[0264] In some embodiments, the regulatory factor comprises a site-specific binding domain, such as a DNA-binding protein or DNA-binding nucleic acid, that specifically binds or hybridizes to a gene at a target region. In some embodiments, the provided polynucleotide or polypeptide is bound or complexed with a site-specific nuclease, such as a modified nuclease. For example, in some embodiments, the administration is carried out using a fusion protein containing a modified nuclease, such as a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, such as a CRISPR-Cas9 system, including an RNA-guided nuclease. In some embodiments, the nuclease is modified to lack nuclease activity. In some embodiments, the modified nuclease is a catalytically dead dCas9.

[0265] In some embodiments, the site-specific binding domain may be derived from a nuclease, for example, a homing enzyme such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. These are recognition sequences for endonucleases and meganucleases. U.S. Patent No. 5,420,032, U.S. Patent No. 6,833,252, Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388, Dujon et al. See also, e.g., Chevalier et al., (1989) Gene 82:115-118; Perler et al., (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. Furthermore, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al., (1996) J. Mol. Biol. 263:163-180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. et al, (2002) Molec. Cell 10:895-905; Epinat et al, (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al, (2006) Nature 441:656-659; Paques et al, (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007 / 0117128.

[0266] Zinc finger, TALE, and CRISPR system binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by manipulating the recognition helix region of naturally occurring zinc finger or TALE proteins (changing one or more amino acids). Engineered DNA binding proteins (zinc finger or TALE) are proteins that do not occur in nature. Rational design criteria include the application of substitution rules and computerized algorithms to process information in databases that store information on existing ZFP and / or TALE designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261; WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496; and U.S. Publication No. 2011 / 0301073.

[0267] In some embodiments, 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. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, and a region of amino acid sequence within the binding domain of that structure is stabilized by the coordination of a zinc ion.

[0268] Among ZFPs are artificial ZFP domains, typically 9–18 nucleotides long, that target specific DNA sequences and are generated by the assembly of individual fingers. ZFPs contain single-finger domains approximately 30 amino acids long, two invariant histidine residues that coordinate zinc with two cysteines in a single beta turn, and an alpha helix with two, three, four, five, or six fingers. Generally, the sequence specificity of ZFPs can be altered by making amino acid substitutions at the four helical positions (−1, 2, 3, and 6) of the zinc finger recognition helix. Thus, in some embodiments, ZFPs or ZFP-containing molecules are engineered to bind to non-naturally occurring, e.g., selected target sites. 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. al. (2001) Curr. Opin. Biotechnol. 12:632-637, Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416, U.S. Patent Nos. 6,453,242, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,030,215, 6,794,136, 7,067,317, 7,262,054, 7,070,934, 7,361,635, 7,253,273, and U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, all of which are incorporated by reference in their entireties.

[0269] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a zinc finger construction platform (CompoZr) in collaboration with Sigma-Aldrich (St. Louis, MO, USA), allowing researchers to completely bypass the construction and validation of zinc fingers and providing zinc fingers specifically targeted to thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom-designed.

[0270] In some embodiments, the site-specific binding domain comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as a transcription activator-like protein effector (TALE) protein. See, e.g., U.S. Patent Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0271] In some embodiments, the site-specific binding domain is derived from a CRISPR / Cas system. Generally, "CRISPR system" collectively refers to transcripts and other factors involved in directing the expression or activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci.

[0272] Generally, the guide sequence comprises a targeting domain comprising a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some examples, the targeting domain of the gRNA is, for example, at least 80, 85, 90, 95, 98, or 99% complementary, for example, fully complementary, to the target sequence on the target nucleic acid.

[0273] In some embodiments, the target site is upstream of the transcription start site of the target gene. In some aspects, the target site is adjacent to the transcription start site of the gene. In some aspects, the target site is adjacent to the RNA polymerase pause site downstream of the transcription start site of the gene.

[0274] In some embodiments, the targeting domain is configured to target the promoter region of the target gene to promote transcription initiation, binding of one or more transcriptional enhancers or activators, and / or RNA polymerase. The target site can be within 600 base pairs on either side of the transcription start site (TSS) of the gene.

[0275] Designing or identifying gRNA sequence is or comprises the sequence of gene targeting, including exon sequence and the sequence of regulatory region including promoter and activator, is within the level of a person skilled in the art.Genome-wide gRNA database for CRISPR genome editing has been published, and it contains exemplary single guide RNA (sgRNA) target sequences in the constitutive exons of genes in human genome or mouse genome (see, for example, genescript.com / gRNA-database.html, and also see Sanjana et al. (2014) Nat.Methods,11:783-4; www.e-crisp.org / E-CRISP / ; crispr.mit.edu / ).In some embodiments, gRNA sequence is or comprises the sequence that has minimal off-target binding to non-target genes.

[0276] In some embodiments, the regulatory element further comprises a functional domain, for example, a transcriptional activator.

[0277] In some embodiments, the transcriptional activator is or includes one or more regulatory factors, such as one or more transcriptional regulators of the target gene, thereby recognizing that the site-specific domains as provided above promote the expression of such genes. In some embodiments, the transcriptional activator promotes the expression of the target gene. In some cases, the transcriptional activator can be or include all or part of a heterologous transactivation domain. For example, in some embodiments, the transcriptional activator is selected from a transactivation domain from herpes simplex, a Dnmt3a methyltransferase domain, p65, VP16, and VP64.

[0278] In some embodiments, the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).

[0279] In certain embodiments, the regulator further comprises a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activator, repressor, coactivator, corepressor), silencer, oncogene (for example, 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 (for example, kinases, acetylases and deacetylases), and DNA modifying enzymes (for example, methyltransferases, such as DNMT family members (for example, DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. For example, see US Publication No. 2013 / 0253040, the entire contents of which are incorporated herein by reference.

[0280] Suitable domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997)), 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. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), as well as degrons (Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplary activation domains include Oct1, Oct-2A, Spl, AP-2, and CTF1 (Seipel et al., EMBO J. 11, 4961-4968 (1992)), as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A, and ERF-2. See, e.g., Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. Steroid. See Biochem. Mol. Biol. 69:3-12, Malik et al. (2000) Trends Biochem. Sci. 25:277-283, and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, AP1, ARF-5, -6, -1, and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1, see, e.g., 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. USA 96:5844-5849, Sprenger-Haussels et al. (2000) Plant J. 22:1-8, Gong et al. See Hobo et al., (1999) Plant Mol. Biol. 41:33-44, and Hobo et al., (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

[0281] Exemplary repression domains that can be used to create gene repressors include, but are not limited to, KRAB A / B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2.See, for example, Bird et al., (1999) Cell 99:451-454, Tyler et al., (1999) Cell 99:443-446, Knoepfler et al., (1999) Cell 99:447-450, and Robertson et al., (2000) Nature Genet. 25:338-342. Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. See Wu et al., (1996) Plant Cell 8:305-321, and Wu et al., (2000) Plant J. 22:19-27.

[0282] In some cases, the domain is involved in epigenetic regulation of chromosomes. In some embodiments, the domain is a nuclear-localized A-type histone acetyltransferase (HAT), such as the MYST family members MOZ, Ybf2 / Sas3, MOF, and Tip60, the 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). In other examples, the domain is a histone deacetylase (HDAC), such as class I (HDAC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7, and 9), HDAC IIB (HDAC 6 and 10)), class IV (HDAC-1 1), or class III (also known as sirtuins (SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898-3941). Another domain used in some embodiments is a histone phosphorylase or kinase, examples of which include MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, Dik / Zip, JAK2, PKC5, WSTF, and CK2. In some embodiments, a methylation domain is used, which may be selected from the group including Ezh2, PRMT1 / 6, PRMT5 / 7, PRMT2 / 6, CARM1, set7 / 9, MLL, ALL-1, Suv39h, G9a, SETDB1, Ezh2, Set2, Dot1, PRMT1 / 6, PRMT5 / 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 (for review, see Kousarides (2007) Cell 128:693-705).

[0283] Fusion molecules are constructed by cloning and biochemical coupling methods well known to those skilled in the art. Fusion molecules contain a DNA binding domain and a functional domain (e.g., a transcription activation or repression domain). Fusion molecules also optionally contain a nuclear localization signal (e.g., from SV40 medium T antigen) and an epitope tag (e.g., FLAG and hemagglutinin). Fusion proteins (and the nucleic acids encoding them) are designed so that the translational reading frame is preserved between the components of the fusion.

[0284] Fusions between the polypeptide component of the functional domain (or functional fragment thereof) on the one hand and the non-protein DNA binding domain (e.g., antibiotics, intercalators, minor groove binders, nucleic acids) on the other hand are constructed by biochemical conjugation methods known to those skilled in the art. See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for making fusions between minor groove binders and polypeptides are described. Mapp et al. al, (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Similarly, CRISPR / Cas TFs and nucleases comprising an sgRNA nucleic acid component in association with a polypeptide component functional domain are also known to those skilled in the art and are described in detail herein.

[0285] The process of introducing the polynucleotide described herein into cells can be achieved by any suitable technique.Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors.In some embodiments, polynucleotides are introduced into cells via viral transduction (for example, lentiviral transduction).

[0286] Once altered, the presence or absence of expression of any of the molecules described herein can be assayed using known techniques such as Western blot, ELISA assay, FACS assay, and the like.

[0287] In some embodiments, the present invention provides hypoimmunogenic pluripotent cells that contain a "suicide gene" or "suicide switch" that is engineered to function as a "safety switch" that can cause death if the hypoimmunogenic pluripotent cells grow and divide in an undesirable manner. The "suicide gene" removal approach can be activated by specific compounds. A suicide gene is included in the gene transfer vector, encoding a protein that causes cell death only when activated. The suicide gene may encode an enzyme that selectively converts non-toxic compounds into highly toxic metabolites, resulting in the specific elimination of cells expressing the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is 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 of which are incorporated herein by reference in their entireties.)

[0288] In other embodiments, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In a preferred embodiment, the inducible caspase protein is iCasp9. This comprises the sequence of the human FK506-binding protein FKBP12 with the F36V mutation connected to the gene encoding human caspase 9 via a series of amino acids. FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903. Therefore, the suicide function of iCasp9 in the present invention is triggered by administration of a dimerization-inducing compound (CID). In some embodiments, the CID is the small molecule drug API903. Dimerization causes rapid induction of apoptosis. (See WO2011 / 146862, Stasi et al, N. Engl. J. Med 365;18 (2011), Tey et al, Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is incorporated by reference in its entirety.)

[0289] L. Generation of Induced Pluripotent Stem Cells The present invention provides a method for producing hypoimmunogenic pluripotent cells. In some embodiments, the method comprises generating pluripotent stem cells. The generation of mouse and human pluripotent stem cells (commonly referred to as iPSCs, miPSCs for mouse cells, or hiPSCs for human cells) is generally known in the art. As will be appreciated by those skilled in the art, there are a variety of different methods for generating iPSCs. Initial induction was performed from mouse embryonic or adult fibroblasts using viral transfer of four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4 (see Takahashi and Yamanaka Cell 126:663-676 (2006), which is incorporated herein by reference in its entirety, particularly for the techniques outlined therein). Since then, many methods have been developed (for a review, see Seki et al. al,World J.Stem Cells 7(1):116-125(2015) and Lakshmipathy and Vermuri,editors,Methods in Molecular Biology:Pluripotent Stem Cells,Methods and Protocols,Springer 2013, both of which are expressly incorporated herein by reference in their entirety, particularly with respect to methods for generating hiPSCs (see, e.g., Chapter 3 of the latter reference).

[0290] Generally, iPSCs are generated by transient expression of one or more reprogramming factors in host cells, usually introduced using an episomal vector. Under these conditions, a small number of cells are induced to become iPSCs (generally, the efficiency of this step is low because no selectable markers are used). Once the cells are "reprogrammed" to pluripotency, they lose the episomal vector and use endogenous genes to produce the factors.

[0291] As will also be appreciated by those skilled in the art, the number of reprogramming factors that can be used or can be employed can vary. Generally, using fewer reprogramming factors will result in less efficient conversion of cells to a pluripotent state, as well as less "pluripotency", e.g., fewer reprogramming factors can result in cells that are not fully pluripotent and can only differentiate into fewer cell types.

[0292] In some embodiments, a single reprogramming factor, OCT4, is used. In other embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other embodiments, three reprogramming factors, OCT4, KLF4, and SOX2, are used. In other embodiments, four reprogramming factors, OCT4, KLF4, SOX2, and c-Myc, are used. In other embodiments, five, six, or seven reprogramming factors selected from SOKMNLT, SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen can be used. Generally, these reprogramming factor genes are provided in episomal vectors, as are known in the art and commercially available.

[0293] Generally, as known in the art, iPSCs are generated from non-pluripotent cells, such as, but not limited to, blood cells, fibroblasts, etc., by transiently expressing reprogramming factors as described herein.

[0294] M. Assays for retention of hypoimmunogenic phenotype and pluripotency Once the hypoimmunogenic cells have been generated, they can be assayed for their hypoimmunogenicity and / or retention of pluripotency as described in WO2016 / 183041 and WO2018 / 132783.

[0295] In some embodiments, low immunogenicity is assayed using several techniques, such as those illustrated in Figures 13 and 15 of WO2018 / 132783. These techniques include transplantation into an allogeneic host and monitoring for hypoimmunogenic pluripotent cell growth (e.g., teratomas) that escape the host's immune system. In some cases, hypoimmunogenic pluripotent cell derivatives are transduced to express luciferase and can then be tracked using bioluminescence imaging. Similarly, the host animal's T cell and / or B cell responses to such cells are tested to ensure that the cells do not elicit an immune reaction in the host animal. T cell function can be assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). B cell or antibody responses are assessed using FACS or Luminex. Additionally or alternatively, cells can be assayed for their ability to evade an innate immune response, e.g., NK cell killing, as generally shown in Figures 14 and 15 of WO2018 / 132783.

[0296] In some embodiments, the immunogenicity of the cells is assessed using T cell immunoassays, such as T cell proliferation assays, T cell activation assays, and T cell killing assays, as recognized by those skilled in the art. In some cases, T cell proliferation assays involve pre-treating cells with interferon-gamma, co-culturing the cells with labeled T cells, and assaying for the presence of a T cell population (or an expanded T cell population) after a preselected time. In some cases, T cell activation assays involve co-culturing T cells with cells as outlined herein and determining the expression level of a T cell activation marker in the T cells.

[0297] To assess the immunogenicity of the cells outlined herein, in vivo assays can be performed. In some embodiments, the survival and immunogenicity of hypoimmunogenic cells are determined using an allogeneic humanized immunodeficient mouse model. In some cases, hypoimmunogenic Pluripotent stem cells are transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell viability, and teratoma formation. In some cases, transplanted, hypoimmunogenic pluripotent stem cells or their differentiated cells show long-term survival in mouse models.

[0298] Additional techniques for determining immunogenicity, including hypoimmunogenicity of cells, are described, for example, in Deuse et al. et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, the disclosures of which, including figures, figure legends, and method descriptions, are incorporated herein by reference in their entirety.

[0299] Similarly, retention of pluripotency is tested in several ways. In one embodiment, pluripotency is assayed by expression of certain pluripotency-specific factors, such as those generally described herein and shown in Figure 29 of WO2018 / 132783. Additionally or alternatively, pluripotent cells are differentiated into one or more cell types as an indicator of pluripotency.

[0300] As will be appreciated by those skilled in the art, successful reduction of MHC1 function (HLA I if the cells are derived from human cells) in pluripotent cells can be measured using the following techniques known in the art, for example, FACS techniques using labeled antibodies that bind to the HLA complex, e.g., using commercially available HLA-A, B, C antibodies that bind to the alpha chain of human major histocompatibility gene HLA class I antigens.

[0301] Additionally, cells can be tested to ensure that HLA-I complexes are not expressed on the cell surface, which can be assayed by FACS analysis using antibodies against one or more HLA cell surface components, as described above.

[0302] Successful reduction of MHC II function (or HLA II if the cells are derived from human cells) in pluripotent cells or their derivatives can be measured using techniques known in the art, such as Western blotting using antibodies against the protein, FACS techniques, RT-PCR techniques, etc.

[0303] Additionally, cells can be tested to ensure that no HLA II complexes are expressed on the cell surface. Again, this assay is performed as known in the art (see, e.g., Figure 21 of WO2018 / 132783) and generally includes human HLA class II complexes. This is done using either Western blot or FACS analysis based on commercially available antibodies that bind to HLA-DR, DP, and most DQ antigens.

[0304] In addition to reduced HLA I and II (or MHC I and II), the hypoimmunogenic cells of the present invention have reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting hypoimmunogenic cells "escape" immune macrophages and innate immune pathways by expressing one or more CD24 transgenes.

[0305] N. Maintenance of Low-Immunogenic Pluripotent Stem Cells Once hypoimmunogenic pluripotent stem cells are generated, they can be maintained in an undifferentiated state, as is known for maintaining iPSC cells. For example, cells can be cultured on Matrigel using a medium that prevents differentiation and maintains pluripotency. Furthermore, they can be placed in a medium under conditions that maintain pluripotency.

[0306] O. Differentiation of Pluripotent Stem Cells The present invention provides pluripotent stem cells that can differentiate into different cell types for subsequent transplantation into a recipient subject. Differentiation is generally determined by assessing the presence of cell-specific markers. As will be appreciated by those skilled in the art, differentiated, hypoimmunogenic pluripotent cell derivatives can be transplanted using techniques known in the art that depend on both the cell type and the ultimate use of these cells.

[0307] 1. Cardiac cells differentiated from pluripotent stem cells The present invention provides pluripotent stem cells that can differentiate into different types of cardiac cells for subsequent transplantation or engraftment into a subject (e.g., a recipient). As will be understood by those skilled in the art, the method for differentiation using known methods depends on the desired type of cell. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodal cardiomyocytes, conductive cardiomyocytes, working cardiomyocytes, cardiomyocyte progenitor cells, cardiac stem cells, atrial cardiac stem cells, ventricular cardiac stem cells, epicardial cells, hematopoietic cells, vascular endothelial cells, endocardial endothelial cells, cardiac valve interstitial cells, cardiac pacemaker cells, and the like.

[0308] In some embodiments, cardiomyocyte precursors include cells that can give rise to progeny, including mature (end-stage) cardiomyocytes (without dedifferentiation or reprogramming). Cardiomyocyte progenitor cells can often be identified using one or more markers selected from GATA-4, Nkx2.5, and the MEF-2 family of transcription factors. In some cases, cardiomyocytes refer to immature or mature cardiomyocytes that express one or more markers (in some cases at least three or five markers) from the following list: cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, β2-adrenergic receptor, ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF). In some embodiments, cardiac cells exhibit spontaneous periodic contractile activity. In some cases, cardiac cells are differentiated by appropriate Ca expression. 2+When cultured in an appropriate tissue culture environment with appropriate concentration and electrolyte balance, the cells can be observed to contract periodically along one axis of the cell and then release from the contraction without the need for additional components in the medium. In some embodiments, the cardiac cells are hypoimmunogenic cardiac cells.

[0309] In some embodiments, the cardiac cells described herein are used to treat a variety of conditions, including pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathies, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, pleural vessels, rheumatic heart disease, arteritis, cardiovascular disease, myocardial infarction, myocardial ischemia, cavities, and the like. The composition is administered to a recipient subject to treat a cardiac disorder selected from the group consisting of congestive heart failure, myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, conduction system dysfunction, coronary artery silk dysfunction, pulmonary hypertension, cardiac arrhythmias, muscular dystrophy, muscle mass abnormalities, muscle degeneration, myocarditis, infectious myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, and autoimmune endocarditis.

[0310] In some embodiments, a method for producing a population of hypoimmunogenic cardiac cells from a population of hypoimmunogenic pluripotent (HIP) cells by in vitro differentiation includes: (a) culturing the population of HIP cells in a culture medium containing a GSK inhibitor; (b) culturing the population of HIP cells in a culture medium containing a WNT antagonist to produce a population of procardial cells; and (c) culturing the population of procardial cells in a culture medium containing insulin to produce a population of hypoimmunogenic cardiac cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. Optionally, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. Optionally, the WNT antagonist is IWR1. The agonist is at a concentration ranging from about 2 mM to about 10 mM.

[0311] In some embodiments, the population of hypoimmunogenic cardiac cells is isolated from non-cardiac cells. In some embodiments, the isolated population of hypoimmunogenic cardiac cells is expanded prior to administration. In certain embodiments, the isolated population of hypoimmunogenic cardiac cells is expanded and cryopreserved prior to administration.

[0312] Other useful methods for differentiating induced pluripotent stem cells or multipotent stem cells into cardiac cells are described, for example, in US2017 / 0152485, US2017 / 0058263, US2017 / 0002325, US2016 / 0362661, US2016 / 0068814, US9,062,289, US7,897,389, and US7,452,718. Additional methods for producing cardiac cells from induced pluripotent stem cells or multipotent stem cells are described, for example, in Xu et al., Stem Cells and Development, 2006, 15(5):631-9, Burridge et al., Cell Stem Cell, 2012, 10:16-28, and Chen et al., Stem Cell Res, 2015, 15(2):365-375.

[0313] In various embodiments, the hypoimmunogenic cardiac cells can be grown in a medium comprising a BMP pathway inhibitor, a WNT signaling activator, a WNT signaling inhibitor, a WNT agonist, a WNT antagonist, a Src inhibitor, an EGFR inhibitor, a PCK activator, a cytokine, a growth factor, a cardiotropic substance, a compound, or the like.

[0314] WNT signaling activators include, but are not limited to, CHIR99021. PCK activators include, but are not limited to, PMA. WNT signaling inhibitors include, but are not limited to, compounds selected from KY02111, SO3031 (KY01-I), SO2031 (KY02-I), and SO3042 (KY03-I), and XAV939. Src inhibitors include, but are not limited to, A41959. EGFR inhibitors include, but are not limited to, AG1478.

[0315] Non-limiting examples of agents for generating cardiac cells from iPSCs include activin A, BMP-4, Wnt3a, VEGF, soluble frizzled proteins, cyclosporine A, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2'-deoxycytidine, and the like.

[0316] The cells of the present invention can be cultured on surfaces, such as synthetic surfaces, to support and / or promote the differentiation of hypoimmunogenic pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymeric material, including, but not limited to, homopolymers or copolymers of one or more selected acrylate monomers. Non-limiting examples of acrylate and methacrylate monomers include tetra(ethylene glycol) diacrylate, glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethylene glycol) dimethacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane epoxide (1EO / QH) methyl, tricyclo[5.2.1.0]dimethacrylate, methyl ... 2,6] decanedimethanol diacrylate, neopentyl glycol ethoxylate diacrylate, and trimethylolpropane triacrylate. Acrylates were synthesized as known in the art or purchased from Polysciences, Inc., Sigma Aldrich. These compounds were obtained from commercial vendors such as Sigma, Inc. and Sartomer, Inc.

[0317] The polymeric material can be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic materials, glass, plastics, polymers or copolymers, any combination thereof, or coatings of one material on another material. In some cases, the glass includes soda-lime glass, Pyrex glass, Vycor glass, quartz glass, silicon, or derivatives thereof.

[0318] In some cases, the plastics or polymers, including dendritic polymers, include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine, or derivatives thereof. In some cases, the copolymers include poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid), or derivatives thereof.

[0319] The efficacy of cardiac cells prepared as described herein can be evaluated in an animal model of cardiac cryoinjury, in which 55% of the left ventricular wall tissue becomes scar tissue without treatment (Li et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999; Sakai et al., Thorac. Cardiovasc. Surg. 118:715, 1999). Successful treatment can reduce the scar area, limit scar expansion, and improve cardiac function, as determined by systolic, diastolic, and generated pressures. Cardiac injury can also be modeled using an embolic coil placed in the distal portion of the left anterior descending artery (Watanabe et al., Cell Transplant. 7:239, 1998), and the efficacy of treatment can be assessed by histology and cardiac function.

[0320] In some embodiments, administration comprises implantation, intravenous injection, intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion into the cardiac tissue of the subject.

[0321] In some embodiments, patients receiving engineered cardiac cells also receive a cardiac medication. Illustrative cardiac medications suitable for use in combination therapy include, but are not limited to, growth factors, polynucleotides encoding growth factors, angiogenic agents, calcium channel blockers, antihypertensives, antimitotics, inotropes, antiatherogenics, anticoagulants, beta-blockers, antiarrhythmics, anti-inflammatory agents, vasodilators, thrombolytics, cardiac glycosides, antibiotics, antivirals, antifungals, agents inhibiting protozoa, nitrates, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, brain natriuretic peptide (BNP), antitumor agents, steroids, and the like.

[0322] The effectiveness of treatment with the methods of the present invention can be monitored in a variety of ways. For example, an electrocardiogram (ECG) or Horrier monitor can be used to determine the effectiveness of treatment. An ECG is a measure of heart rhythm and electrical impulses and is a highly effective and non-invasive method for determining whether a treatment has improved, maintained, prevented, or delayed the decline in electrical conduction in a subject's heart. The use of a Horrier monitor, a portable ECG that can be worn over long periods of time to monitor cardiac abnormalities and arrhythmia disorders, is also a reliable method for assessing the effectiveness of treatment. An ECG or nuclear medicine scan can be used to determine improvement in ventricular function.

[0323] 2. Neurons differentiated from pluripotent stem cells The present invention provides pluripotent stem cells that can differentiate into different types of neural cells for subsequent transplantation or engraftment into recipient subjects.As will be understood by those skilled in the art, the method for differentiation using known methods depends on the desired type of cells.Exemplary types of neural cells include, but are not limited to, brain endothelial cells, neurons, glial cells, etc.

[0324] In some embodiments, neural cells are administered to a subject to treat Parkinson's disease, Huntington's disease, multiple sclerosis, other neurodegenerative diseases or conditions, attention deficit hyperactivity disorder (ADHD), Tourette's syndrome (TS), schizophrenia, psychosis, depression, or other neuropsychiatric disorders. In some embodiments, neural cells described herein are administered to a subject to treat or ameliorate a stroke. In some embodiments, neurons and glial cells are administered to a subject with amyotrophic lateral sclerosis (ALS). In some embodiments, brain endothelial cells are administered to reduce the symptoms or effects of a cerebral hemorrhage. In some embodiments, dopaminergic neurons are administered to a patient with Parkinson's disease. In some embodiments, noradrenergic neurons and GABAergic interneurons are administered to a patient experiencing an epileptic seizure. In some embodiments, motor neurons, interneurons, Schwann cells, oligodendrocytes, and microglia are administered to a patient experiencing a spinal cord injury.

[0325] In some embodiments, brain endothelial cells (ECs), their precursors, and their progenitor cells are differentiated from pluripotent stem cells (e.g., induced pluripotent stem cells) on a surface by culturing the cells in a medium containing one or more factors that promote the generation of brain ECs or neural cells. In some embodiments, the medium contains one or more of the following: CHIR-99021, VEGF, basic FGF, and Y-27632. In some embodiments, the medium contains supplements designed to promote the survival and functionality of neural cells.

[0326] In some embodiments, brain endothelial cells (ECs), precursors, and their progenitors are differentiated from pluripotent stem cells on a surface by culturing the cells in unconditioned or conditioned medium. In some cases, the medium contains factors or small molecules that promote or facilitate differentiation. In some embodiments, the medium contains one or more factors or small molecules selected from the group consisting of VEGR, FGF, SDF-1, CHIR-99021, Y-27632, SB 431542, and any combination thereof. In some embodiments, the surface for differentiation contains one or more extracellular matrix proteins. The surface can be coated with one or more extracellular matrix proteins. Cells are differentiated in suspension into a gel matrix, such as Matrigel, gelatin, or fibrin / thrombin, which promotes cell survival. In some cases, differentiation can be assayed, as known in the art, generally by assessing the presence of cell-specific markers.

[0327] In some embodiments, the brain endothelial cells express or secrete a factor selected from the group consisting of CD31, VE-cadherin, and combinations thereof. In certain embodiments, the brain endothelial cells express or secrete a factor selected from the group consisting of CD31, CD34, CD45, CD117 (c-kit), CD146, CXCR4, VEGF, SDF-1, PDGF, GLUT-1, PECAM-1, eNOS, claudin-5, occludin, ZO-1, p-glycoprotein, von Willebrand factor, VE-cadherin, low-density lipoprotein receptor LDLR, low-density lipoprotein receptor-related protein 1 LRP1, insulin receptor INSR, leptin receptor LEPR, basal cell adhesion molecule BCAM, transferrin receptor TFRC, advanced glycation end products-specific receptor AGER, retinol uptake receptor STRA6, large neutral amino acid transporter small subunit 1 SLC7A5, and excitatory amino acid transporter 3 SL. and expressing or secreting one or more factors selected from the group consisting of C1A1, sodium-binding neutral amino acid transporter 5 SLC38A5, solute carrier family 16 member 1 SLC16A1, ATP-dependent translocase ABCB1, ATP-ABCC2-binding cassette transporter ABCG2, multidrug resistance-associated protein 1 ABCC1, canalicular multispecific organic anion transporter 1 ABCC2, multidrug resistance-associated protein 4 ABCC4, and multidrug resistance-associated protein 5 ABCC5.

[0328] In some embodiments, the brain ECs are characterized by one or more features selected from the group consisting of high expression of tight junctions, high electrical resistance, low fenestrations, small perivascular spaces, high prevalence of insulin and transferrin receptors, and large numbers of mitochondria.

[0329] In some embodiments, brain ECs are selected or purified using a positive selection strategy. In some cases, brain ECs are sorted for endothelial cell markers, such as, but not limited to, CD31. In other words, CD31-positive brain ECs are isolated. In some embodiments, brain ECs are selected or purified using a negative selection strategy. In some embodiments, undifferentiated or pluripotent stem cells are removed by selecting cells that express pluripotency markers, including, but not limited to, TRA-1-60 and SSEA-1.

[0330] In some embodiments, neurons, their precursors and progenitor cells are differentiated from pluripotent stem cells by culturing the cells in a medium containing one or more factors selected from the group consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, a SMAD inhibitor, a Wnt antagonist, an activator of SHH signaling, and any combination thereof. In some embodiments, the SMAD inhibitor is SB431542, LDN-193189, noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, meterimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, K 26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, and derivatives thereof. In some embodiments, the Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, DKK-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, and derivatives thereof. In some embodiments, the SHH signaling activator is selected from the group consisting of smoothened agonist (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, and derivatives thereof.

[0331] In some embodiments, the neuron is a member of the glutamate ionotropic receptor NMDA-type subunit 1 GRIN1, glutamic acid decarboxylase 1 GAD1, gamma aminobutyric acid GABA, tyrosine hydroxylase TH, LIM homeobox transcription factor 1-alpha LMX1A, forkhead box protein O1 FOXO1, forkhead box protein A2 FOXA2, forkhead box protein O4 FOXO4, FOXG1, 2',3'-cyclic-nucleotide 3'-phosphodiesterase CNP, myelin basic protein MBP, tubulin beta chain 3 TUB3, tubulin beta chain 3 NEUN, solute carrier family 1 member 6 SLC1A6, SST, PV, calbindin, RAX, LHX6, LHX8, DLX1, DLX2, D In some embodiments, the dopaminergic neurons express one or more markers selected from the group consisting of LX5, DLX6, SOX6, MAFB, NPAS1, ASCL1, SIX6, OLIG2, NKX2.1, NKX2.2, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, ChAT, NGFI-B, c-fos, CRF, RAX, POMC, hypocretin, NADPH, NGF, Ach, VAChT, PAX6, EMX2p75, CORIN, TUJ1, NURR1, and any combination thereof. In some embodiments, the dopaminergic neurons express one or more markers selected from CORIN, FOXA2, TUJ1, NURR1, and any combination thereof.

[0332] In some embodiments, the stem cells described herein are differentiated into dopaminergic neurons and contain dopaminergic progenitor cells. The stem cells are cultured in a differentiation medium containing supplementary substances or additives to induce neural differentiation. In some embodiments, the cells are cultured in the presence of supplementary substances or additives to induce floor plate cells. In some embodiments, the supplementary substances or additives include the BMP inhibitor LDN193189, the ALK-5 inhibitor A83-01, the smoothened agonist purmorphamine, FGF8, the GSK3 inhibitor CHIR99021, glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, brain-derived neurotrophic factor (BDNF), dibutyryl adenosine cyclic monophosphate (dbcAMP), and the ROCK inhibitor Y-27632.

[0333] In some embodiments, a method for producing a population of hypoimmunogenic dopaminergic neurons from a population of hypoimmunogenic induced pluripotent stem cells (HIP cells) by in vitro differentiation includes: (a) culturing the population of HIP cells in a first culture medium containing one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3 inhibitor, an ALK inhibitor, and a ROCK inhibitor to produce a population of immature dopaminergic neurons; and (b) culturing the population of immature dopaminergic neurons in a second culture medium different from the first culture medium to produce a population of dopaminergic neurons. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. Optionally, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the ALK inhibitor is at a concentration ranging from about 1 mM to about 10 mM. In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

[0334] In some embodiments, the population of hypoimmunogenic dopaminergic neurons is isolated from non-neuronal cells. In some embodiments, the isolated population of hypoimmunogenic dopaminergic neurons is expanded prior to administration. In certain embodiments, the isolated population of hypoimmunogenic dopaminergic neurons is expanded and cryopreserved prior to administration.

[0335] Methods for differentiating pluripotent stem cells are described, for example, in Kikuchi et al., Nature, 2017, 548, 592-596; Kriks et al., Nature, 2011, 547-551; Doi et al., Stem Cell Reports, 2014, 2, 337-50; Perrier et al., Proc Natl Acad Sci USA, 2004, 101, 12543-12548; Chambers et al., Nat Biotechnol, 2009, 27, 275-280; and Kirkeby et al., Cell Reports, 2012, 1, 703-714.

[0336] Useful descriptions of stem cell-derived neurons and methods for their generation can be found in, for example, Kirkeby et al., Cell Rep, 2012, 1:703-714; Kriks et al., Cell Rep, 2012, 1:703-714; et al.,Nature,2011,480:547-551, Wang et al.,Stem Cell Reports,2018,11(1):171-182, Lorenz Studer, "Chapter 8-Strategies for Bringing Stem Cell-Derived Dopamine Neurons to the clinic-The NYSTEM Trial" in Progress in Brain Research,2017,volume 230, pg.191-212, Liu et al., Nat Protoc,2013,8:1670-1679, Upadhya et al., Curr Protoc Stem Cell Biol, 38, 2D.7.1-2D.7.47, U.S. Published Application No. 2016 / 0115448, and US Pat. No. 8,252,586, US Pat. No. 8,273,570, US Pat. No. 9,487,752 and US Pat. No. 10,093,897, the contents of which are incorporated herein by reference in their entireties.

[0337] In some embodiments, glial cells, including microglia, astrocytes, oligodendrocytes, ependymal cells, and Schwann cells, their glial precursors, and glial progenitor cells are produced by differentiating pluripotent stem cells into therapeutically effective glial cells, etc. Differentiation of hypoimmunogenic pluripotent stem cells produces hypoimmunogenic neural cells, such as hypoimmunogenic glial cells.

[0338] In some embodiments, glial cells, their precursors, and progenitor cells are generated by culturing pluripotent stem cells in a medium containing one or more agents selected from the group consisting of retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, TGF-beta inhibitor, BMP signaling inhibitor, SHH signaling activator, FGF, platelet-derived growth factor PDGF, PDGFR-alpha, HGF, IGF-1, noggin, sonic hedgehog (SHH), dorsomorphin, noggin, and any combination thereof. In certain examples, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof. In some embodiments, the glial cells express NKX2.2, PAX6, SOX10, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3, NT-4), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), FGF8, EGFR, OLIG1, OLIG2, myelin basic protein (MBP), GAP-43, LNGFR, nestin, GFAP, CD11b, CD11c, CX3CR1, P2RY12, IBA-1, TMEM119, CD45, and any combination thereof. Exemplary differentiation media can include any specific factors and / or small molecules that can promote or enable the generation of glial cell types recognized by those skilled in the art.

[0339] To determine whether cells generated according to an in vitro differentiation protocol exhibit glial cell characteristics and properties, the cells can be transplanted into an animal model. In some embodiments, glial cells are injected into immunodeficient mice, such as immunodeficient Shiverer mice. The glial cells are administered into the brain of the mouse, and after a preselected time, the transplanted cells are evaluated. In some cases, the cells engrafted into the brain are visualized using immunostaining and imaging methods. In some embodiments, it is determined that the glial cells express known glial cell biomarkers.

[0340] Useful methods for generating glial cells, progenitor cells, and their precursors from stem cells can be found, for example, in US 7,579,188, US 7,595,194, US 8,263,402, US 8,206,699, US 8,252,586, US 9,193,951, US 9,862,925, US 8,227,247, US 9,709,553, US 2018 / 0187148, US 2017 / 0198255, US 2017 / 0183627, US 2017 / 0182097, US 2017 / 253856, US 2018 / 0236004, WO 2017 / 172976, and WO 2018 / 093681.

[0341] In some embodiments, differentiation of pluripotent stem cells is carried out by exposing or contacting the cells with specific factors known to produce specific cell lineages in order to target their differentiation to specific, desired lineages and / or cell types of interest. In some embodiments, terminally differentiated cells exhibit specialized phenotypic properties or characteristics. In certain embodiments, the stem cells described herein differentiate into neuroectodermal, neural, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic neurons, parasympathetic neurons, sympathetic peripheral neurons, or glial cell populations. In some cases, the glial cell population includes microglial (e.g., amoeboid, ramified, activated phagocytes, and activated non-phagocytes) cell populations or macroglial (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells, and radial glia; and peripheral nervous system cells: Schwann cells and satellite cells) cell populations, or precursors and progenitors of any of the preceding cells.

[0342] Protocols for generating different types of neural cells are described in PCT Application No. 2010 / 144696, U.S. Patent Nos. 9,057,053, 9,376,664, and 10,233,422. Additional descriptions of methods for differentiating hypoimmunogenic pluripotent cells can be found, for example, in Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc. Methods for determining the effects of neural cell transplantation in animal models of neurological disorders or conditions are described in the following references: for spinal cord injury—Curtis et al., Cell Stem Cell, 2018, 22, 941-950; for Parkinson's disease—Kikuchi et al., Nature, 2017, 548:592-596; for ALS—Izrael et al., Stem Cell Research, 2018, 9(1):152 and Izrael et al., IntechOpen, DOI:10.5772 / intechopen.72862; for epilepsy—Upadhya et al., PNAS, 2019, 116(1):287-296.

[0343] The efficacy of neural cell transplants for spinal cord injury can be evaluated in rat models of acute spinal cord injury, as described, for example, by McDonald, et al., Nat. Med., 1999, 5:1410 and Kim, et al., Nature, 2002, 418:50. For example, if the transplant is successful, transplant-derived cells may be found to reside in the lesion after 2–5 weeks, differentiate into astrocytes, oligodendrocytes, and / or neurons, migrate from the lesion edge along the spinal cord, and improve locomotion, coordination, and weight bearing. A specific animal model is selected based on the type of neural cell and the neurological disease or condition being treated.

[0344] Neuronal cells can be administered in a manner that allows them to engraft at the intended tissue site and reconstitute or regenerate functionally deficient areas. For example, neuronal cells can be directly transplanted into the parenchyma or intrathecal site of the central nervous system, depending on the disease being treated. In some embodiments, any of the neuronal cells described herein, including brain endothelial cells, neurons, dopaminergic neurons, ependymal cells, astrocytes, microglial cells, oligodendrocytes, and Schwann cells, are injected into a patient intravenously, intraspinally, intracerebroventricularly, intrathecally, intraarterially, intramuscularly, intraperitoneally, subcutaneously, intramuscularly, intraperitoneally, intraocularly, retrobulbarly, and combinations thereof. In some embodiments, the cells are injected or deposited in the form of a bolus injection or continuous infusion. In certain embodiments, the neuronal cells are administered by injection into the brain, by being appropriately positioned in the brain, or combinations thereof. Injection is For example, administration can be performed through a burr hole made in the subject's skull. Suitable sites for administering neurons to the brain include, but are not limited to, the ventricles, lateral ventricles, cisterna magna, putamen, basal ganglia, hippocampal cortex, striatum, caudate region of the brain, and combinations thereof.

[0345] Additional description of neural cells, including dopaminergic neurons, for use in the present invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0346] 3. Endothelial cells differentiated from pluripotent stem cells The present invention provides pluripotent stem cells that can differentiate into different types of endothelial cells for subsequent transplantation or engraftment into a subject (e.g., a recipient). As will be understood by those skilled in the art, the method for differentiation using known methods depends on the desired type of cells. Exemplary types of endothelial cells include, but are not limited to, capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, arterial endothelial cells, venous endothelial cells, kidney endothelial cells, brain endothelial cells, liver endothelial cells, etc.

[0347] The endothelial cells outlined herein can express one or more endothelial cell markers. Non-limiting examples of such markers include VE-cadherin (CD144), ACE (angiotensin-converting enzyme) (CD143), BNH9 / BNF13, CD31, CD34, CD54 (ICAM-1), CD62E (E-selectin), CD105 (endoglin), CD146, endocan (ESM-I), endoglix-I, endomucin, eotaxin-3, EPAS1 (endothelial PAS domain protein 1), factor VIII-related antigen, FLI-1, Flk-1 (KDR, VEGFR-2), FLT-1 (VEGFR-1), GATA2, GBP-1 (guanylate-binding protein-1), GRO-alpha, HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, LYVE-1, MRB (magic roundabout), nucleolin, PAL-E (pathologische Anatomie Leiden-endothelial), RTK, sVCAM-1, TALI, TEM1 (tumor endothelial marker 1), TEM5 (tumor endothelial marker 5), TEM7 (tumor endothelial marker 7), thrombomodulin (TM, CD141), VCAM-1 (vascular cell adhesion molecule-1) (CD106), VEGF (vascular endothelial growth factor), vWF (von Willebrand factor), ZO-1, endothelial cell-selective adhesion molecule (ESAM), CD102, CD93, CD184, CD304, and DLL4.

[0348] In some embodiments, the endothelial cells are genetically modified to express an exogenous gene encoding a protein of interest, such as, but not limited to, an enzyme, hormone, receptor, ligand, or drug useful for treating a disorder / condition or ameliorating the symptoms of a disorder / condition. Standard methods for genetically modifying endothelial cells are described, for example, in US 5,674,722.

[0349] Such endothelial cells can be used to provide constitutive synthesis and delivery of polypeptides or proteins useful for the prevention or treatment of disease. In this way, the polypeptides are secreted directly into the individual's bloodstream or other areas of the body (e.g., the central nervous system). In some embodiments, endothelial cells can be modified to secrete insulin, blood clotting factors (e.g., factor VIII or von Willebrand factor), alpha-1 antitrypsin, adenosine deaminase, tissue plasminogen activator, interleukins (e.g., IL-1, IL-2, IL-3), and the like.

[0350] In certain embodiments, endothelial cells may be modified in ways that improve their performance in the context of an implanted graft. Non-limiting examples include secreting or expressing thrombolytic agents to prevent intraluminal clot formation, smooth muscle hypertrophy to prevent luminal narrowing due to smooth muscle hypertrophy, and the like. These include the secretion of inhibitors of muscle proliferation, and the expression and / or secretion of endothelial cell mitogens or autocrine factors to stimulate endothelial cell proliferation and improve the extent or duration of endothelial cell lining of the graft lumen.

[0351] In some embodiments, engineered endothelial cells are utilized to deliver therapeutic levels of secreted products to specific organs or limbs. For example, vascular implants lined with in vitro engineered (transduced) endothelial cells can be implanted into specific organs or limbs. The secreted products of the transduced endothelial cells are delivered in high concentrations to the perfused tissue, thereby achieving the desired effect at the targeted anatomical location.

[0352] In other embodiments, the endothelial cells are genetically modified to contain a gene that disrupts or inhibits angiogenesis when expressed by the endothelial cells in angiogenic tumors. In some cases, the endothelial cells may also be genetically modified to express any one of the selectable suicide genes described herein, which allows for negative selection of transplanted endothelial cells upon completion of tumor treatment.

[0353] In some embodiments, the endothelial cells described herein are administered to a recipient subject to treat a vascular disorder selected from the group consisting of vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, hypertension, ischemic tissue damage, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, renal failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, peritoneal and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal artery stenosis, obstruction of the lower limbs, and other vascular conditions or diseases.

[0354] In some embodiments, the hypoimmunogenic pluripotent cells are differentiated into endothelial colony-forming cells (ECFCs) to form new blood vessels to treat peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., doi:10.1038 / nbt.3048, which is incorporated herein by reference in its entirety, particularly for methods and reagents for generating endothelial cells from human pluripotent stem cells, and for transplantation techniques. Differentiation can generally be assayed as known in the art by assessing the presence of endothelial cell-associated or specific markers, or by functional measurement.

[0355] In some embodiments, a method for producing a population of hypoimmunogenic endothelial cells from a population of hypoimmunogenic pluripotent cells by in vitro differentiation comprises: (a) culturing a population of HIP cells in a first culture medium comprising a GSK inhibitor; (b) culturing the population of HIP cells in a second culture medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre-endothelial cells in a third culture medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of hypoimmunogenic endothelial cells.

[0356] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. Optionally, the GSK inhibitor is at a concentration ranging from about 1 mM to about 10 mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. Optionally, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 20 pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. Optionally, the ALK inhibitor is at a concentration ranging from about 0.5 pM to about 10 pM.

[0357] In some embodiments, the first culture medium contains between 2 pM and about 10 pM CHIR-99 021. In some embodiments, the second culture medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 pM Y-27632 and 1 pM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In certain examples, the first culture medium and / or the second culture medium do not comprise insulin.

[0358] The cells of the present invention can be cultured on surfaces, such as synthetic surfaces, to support and / or promote the differentiation of hypoimmunogenic pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymeric material, including, but not limited to, homopolymers or copolymers of one or more selected acrylate monomers. Non-limiting examples of acrylate and methacrylate monomers include tetra(ethylene glycol) diacrylate, glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethylene glycol) dimethacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane epoxide (1EO / QH) methyl, tricyclo[5.2.1.0]dimethacrylate, methyl ... 2,6 ] decanedimethanol diacrylate, neopentyl glycol exoxylate diacrylate, and trimethylolpropane triacrylate. Acrylates were synthesized as known in the art or obtained from commercial vendors such as Polysciences, Inc., Sigma Aldrich, Inc., and Sartomer, Inc.

[0359] In some embodiments, endothelial cells can be seeded onto a polymer matrix. In some cases, the polymer matrix is ​​biodegradable. Suitable biodegradable matrices are well known in the art and include collagen-GAG, collagen, fibrin, PLA, PGA, and PLA / PGA copolymers. Additional biodegradable materials include poly(anhydrides), poly(hydroxy acids), poly(orthoesters), poly(propyl fumerate), poly(caprolactone), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes, and polysaccharides.

[0360] Non-biodegradable polymers can also be used. Other non-biodegradable but biocompatible polymers include polypyrrole, polyaniline, polythiophene, polystyrene, polyester, non-biodegradable polyurethane, polyurea, poly(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonate, and poly(ethylene oxide). The polymer matrix can be formed into any shape, such as particles, sponges, tubes, spheres, strands, coiled strands, capillary networks, films, fibers, meshes, or sheets. The polymer matrix can be modified to include natural or synthetic extracellular matrix materials and factors.

[0361] The polymeric material can be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic materials, glass, plastics, polymers or copolymers, any combination thereof, or coatings of one material on another material. In some cases, the glass includes soda-lime glass, Pyrex glass, Vycor glass, quartz glass, silicon, or derivatives thereof.

[0362] In some cases, plastics or polymers containing dendritic polymers include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefins, Examples of suitable copolymers include poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid), and derivatives thereof.

[0363] In some embodiments, the population of hypoimmunogenic endothelial cells is isolated from non-endothelial cells. In some embodiments, the isolated population of hypoimmunogenic endothelial cells is expanded prior to administration. In certain embodiments, the isolated population of hypoimmunogenic endothelial cells is expanded and cryopreserved prior to administration.

[0364] Further description of endothelial cells for use in the present invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0365] 4. Thyroid cells differentiated from pluripotent stem cells In some embodiments, pluripotent stem cells can be differentiated into thyroid progenitor cells and thyroid follicular organelles that can secrete thyroid hormone to combat autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art. For example, see Kurmann et al., Cell Stem Cell, 2015 Nov 5:17(5)527-42, the entire contents of which are incorporated herein by reference, particularly for methods and reagents for generating thyroid cells from human pluripotent stem cells, and for transplantation techniques. Differentiation can generally be assayed as known in the art by assessing the presence of thyroid cell-related or specific markers, or by functional measurement.

[0366] 5. Hepatocytes differentiated from pluripotent stem cells In some embodiments, pluripotent stem cells can be differentiated into hepatocytes to address loss of hepatocyte function or cirrhosis. There are many techniques that can be used to differentiate HIP cells into hepatocytes, for example, Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al., Hepatology 51:297-305 (2010), and Asgari et al., Stem See Cell Rev (:493-504 (2013), all of which are incorporated herein by reference in their entireties, particularly with respect to methodologies and reagents for differentiation. Differentiation is generally assayed as known in the art by assessing the presence of hepatocyte-associated and / or specific markers, including, but not limited to, albumin, alpha-fetoprotein, and fibrinogen. Differentiation can also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage.

[0367] 6. Pancreatic islet cells differentiated from pluripotent stem cells The present invention provides pluripotent stem cells that can differentiate into different types of pancreatic islet cells for subsequent transplantation or engraftment into a subject (e.g., a recipient). As will be understood by those skilled in the art, the method for differentiation using known methods will depend on the desired type of cells. Exemplary types of pancreatic islet cells include, but are not limited to, pancreatic islet progenitor cells, immature pancreatic islet cells, mature pancreatic islet cells, and the like. In some embodiments, the pancreatic cells described herein are administered to a subject to treat diabetes.

[0368] In some embodiments, the pancreatic islet cells are derived from the hypoimmunogenic pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into pancreatic islet cells are described, for example, in US 9,683,215, US 9,157,062, and US 8,927,280. There are.

[0369] In some embodiments, the pancreatic islet cells produced by the methods disclosed herein secrete insulin, and in some embodiments, the pancreatic islet cells exhibit at least two characteristics of endogenous pancreatic islet cells, such as, but not limited to, the secretion of insulin in response to glucose and the expression of beta cell markers.

[0370] Exemplary beta cell or beta cell precursor 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.

[0371] In some embodiments, the isolated pancreatic islet cells produce insulin in response to increased glucose. In various embodiments, the isolated pancreatic islet cells secrete insulin in response to increased glucose. In some embodiments, the cells have a distinct morphology, such as a cobblestone cell morphology and / or a diameter of about 17 pm to about 25 pm.

[0372] In some embodiments, the low immunogenicity pluripotent cells are differentiated into beta-like cells or pancreatic islet organoids for transplantation to treat type I diabetes mellitus (T1DM).Cell lines are a promising method for treating T1DM, for example, see Ellis et al., Nat Rev Gastroenterol Hepatol.2017 Oct;14(10):612-628, which is incorporated herein by reference.In addition, Pagliuca et al. (Cell,2014,159(2):428-39) have reported the successful differentiation of beta cells from hiPSCs (the contents of which are incorporated herein in their entirety, particularly for the methods and reagents outlined therein for producing functional human beta cells on a large scale from human pluripotent stem cells). Additionally, Vegas et al. demonstrate the production of human beta cells from human pluripotent stem cells followed by encapsulation to avoid immune rejection by the host (Vegas et al., Nat Med, 2016 22(3):306-11, which is incorporated herein in its entirety, particularly with respect to the methods and reagents outlined therein for large-scale production of functional human beta cells from human pluripotent stem cells).

[0373] In some embodiments, a method for producing a population of hypoimmunogenic pancreatic islet cells from a population of hypoimmunogenic pluripotent cells by in vitro differentiation comprises: (a) culturing a population of HIP cells in a first culture medium containing one or more factors selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-b (TORb) superfamily, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-7 (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 different from the first culture medium, to produce a population of hypoimmunogenic pancreatic islet cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. Optionally, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. Optionally, the ALK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the first culture medium and / or the second culture medium is a medium containing at least one of: Contains no serum.

[0374] In some embodiments, the population of hypoimmunogenic pancreatic islet cells is isolated from non-pancreatic islet cells. In some embodiments, the isolated population of hypoimmunogenic pancreatic islet cells is expanded prior to administration. In certain embodiments, the isolated population of hypoimmunogenic pancreatic islet cells is expanded and cryopreserved prior to administration.

[0375] Differentiation is generally assayed as known in the art by assessing the presence of beta cell-associated or specific markers, including, but not limited to, insulin. Differentiation can also be measured functionally, such as by measuring glucose metabolism; see Muraro et al., Cell Syst. 2016 Oct 26;3(4):385-394.e3, which is incorporated herein by reference in its entirety, particularly for the biomarkers outlined therein. Once beta cells are generated, they can be transplanted into the portal vein / liver, peritoneum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous pouch (either as a cell suspension or within a gel matrix, as discussed herein).

[0376] Further description of pancreatic islet cells containing dopaminergic neurons for use in the present invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0377] 7. Retinal pigment epithelial (RPE) cells differentiated from pluripotent stem cells The present invention provides low immunogenic pluripotent stem cells that can be differentiated into different types of RPE cells for subsequent transplantation or engraftment into a subject (e.g., a recipient).As will be understood by those skilled in the art, the method for differentiation using known methods depends on the desired type of cells.Exemplary types of RPE cells include, but are not limited to, retinal pigment epithelial (RPE) cells, RPE progenitor cells, immature RPE cells, mature RPE cells, functional RPE cells, etc.

[0378] Useful methods for differentiating pluripotent stem cells into RPE cells are described, for example, in US Pat. No. 9,458,428 and US Pat. No. 9,850,463, the disclosures of which, including the specifications, are incorporated herein by reference in their entireties. Additional methods for producing human induced pluripotent stem cells or RPE cells can be found, for example, in Lamba et al., PNAS, 2006, 103(34):12769-12774; Mellough et al., Stem Cells, 2012, 30(4):673-686; Idelson et al., Cell Stem Cell, 2009, 5(4):396-408; Rowland et al., Journal of Cellular Physiology, 2012, 227(2):457-466; Buchholz et al., Stem Cells Trans Med, 2013, 2(5):384-393; and da Cruz et al., Nat Biotech, 2018, 36:328-337.

[0379] In some embodiments, the RPE cells described herein are administered to a subject to treat an ocular disorder selected from the group consisting of wet macular degeneration, dry macular degeneration, early-onset macular degeneration (e.g., Stargardt's disease, Best's disease, and juvenile retinitis pigmentosa), Leber's congenital amaurosis, retinitis pigmentosa, retinal detachment, age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, non-neovascular age-related macular degeneration, and the like.

[0380] Human pluripotent stem cells can be cultured in vitro using techniques outlined in Stem Cell Reports 2014:2:205-18 (which is incorporated herein by reference in its entirety, particularly for the methods and reagents outlined therein regarding differentiation techniques and reagents). The RPE cells are differentiated into RPE cells, see also Mandai et al., N Engl J Med, 2017, 376:1038-1046 (the contents of which are incorporated herein in their entirety for techniques for generating sheets of RPE cells and their transplantation into patients). Differentiation can generally be assayed as known in the art by assessing the presence of RPE cell-associated and / or specific markers or by functional measurement. See, e.g., Kamao et al., Stem Cell Report, 2014, 2(2):205-18, the contents of which are incorporated herein by reference in their entirety, particularly with respect to the markers outlined in the first paragraph of the Results section.

[0381] In some embodiments, a method for producing a population of hypoimmunogenic retinal pigment epithelial (RPE) cells from a population of hypoimmunogenic pluripotent cells by in vitro differentiation includes: (a) culturing the population of hypoimmunogenic pluripotent cells in a first culture medium containing any one of factors selected from the group consisting of activin A, bFGF, BMP4 / 7, DKK1, IGF1, noggin, a BMP inhibitor, an ALK inhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second culture medium different from the first culture medium to produce a population of hypoimmunogenic RPE cells. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. Optionally, the ALK inhibitor is at a concentration ranging from about 2 mM to about 10 pM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 10 pM.In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

[0382] Differentiation can generally be assayed as known in the art by assessing the presence of RPE cell-associated and / or specific markers or by functional measurement, see, e.g., Kamao et al., Stem Cell Differentiation. Report, 2014, 2(2):205-18, the contents of which are incorporated herein by reference in their entirety, particularly with respect to the Results section.

[0383] Further description of RPE cells for use in the present invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0384] For therapeutic use, cells prepared according to the disclosed methods can typically be provided in the form of a pharmaceutical composition containing an isotonic excipient and prepared under conditions sufficiently sterile for human administration. For general principles of pharmaceutical formulation of cell compositions, see "Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy," by Morstyn & Sheridan eds, Cambridge University Press, 1996, and "Hematopoietic Stem Cell Therapy," E.D. Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. Cells can be packaged in devices or containers suitable for distribution or clinical use.

[0385] P. Cell Administration As will be understood by those skilled in the art, differentiated, low-immunogenic pluripotent cell derivatives can be transplanted using techniques known in the art, depending on both the cell type and the ultimate use of these cells. Generally, the cells of the present invention can be transplanted either intravenously or by injection into a specific location in a patient. When transplanted into a specific location, the cells can be suspended in a gel matrix to prevent dispersion while the cells are retained. [Example]

[0386] IV. Working Examples Example 1 The effect of CD24 on macrophage phagocytosis of CD24-expressing cells is measured using the XCELLIGENCE assay. Briefly, human B2M cells transduced with or without a lentiviral vector expressing CD24 (CD24tg) were cultured. - / - CIITA - / -iPSCs were cultured on 10 cm dishes coated with diluted feeder-free Matrigel (hESC Establishment, BD Biosciences, San Jose, CA) in Essential 8 Flex medium (Thermo Fisher Scientific). The medium was changed every 24 hours, and Versene (Gibco) was used for cell passage at a ratio of 1:6. Endothelial cell differentiation began at 60% confluence, when the medium was changed to RPM1-1640 containing 2% B-27 minus insulin (both Gibco) and 5 μM CHIR-99021 (Selleckchem). On day 2, the medium was changed to reduced medium: RPM1-1640 containing 2% B-27 minus insulin and 2 μM CHIR-99021. From day 4 to day 7 of culture, cells were exposed to RPM1-1640 containing RPM1-1640 EC medium, 2% B-27-insulin plus 50 ng / ml human vascular endothelial growth factor (VEGF; R&D Systems), 10 ng / ml human fibroblast growth factor basic (FGFb, R&D Systems), 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542 (Sigma-Aldrich). Endothelial cell clusters are visible from day 7 onwards, and cells are maintained in Endothelial Cell Basal Medium 2 (PromoCell, Heidelberg, Germany) supplemented with the following supplements: 10% FCS hi (Gibco), 1% pen / strep, 25 ng / ml VEGF, 2 ng / ml FGFb, 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542 (Sigma-Aldrich). The differentiation protocol is complete after 14 days, and undifferentiated cells are detached during the differentiation process. TRYPLE EXPRESS (Gibco) is used to passage the cells 1:3 every 3-4 days.

[0387] NK cell killing and macrophage killing assays are performed on the XCELLIGENCE MP platform (ACEA BioSciences). Special 96-well E-plates are coated with gelatin (Millipore) and 4 × 105 B2M - / - CIITA - / - CD24 tg or 4 × 10 5 B2M - / - CIITA - / - hiECs were seeded in 100 μl of cell-specific medium. After the cell index reached 0.7, human NK cells or macrophages were added at a 1:1 effector to target cell (E:T) ratio using 1 μg / ml human IL-2 (PeproTech). As a negative control, cells were treated with 2% Triton X-100. Data were normalized and analyzed using RTCA software (ACEA BioSciences). B2M - / - CIITA - / - hiECs (without CD24) are effectively killed by NK cells and macrophages, but B2M - / - CIITA - / - CD24 tg hiECs are protected from killing by macrophages. Blockade with 10 μg / ml of anti-CD24 antibody (Clone SN3, Novus Biologics) eliminated the protective effect. Overexpression of both CD47 and CD24 is associated with B2M - / - CIITA - / - Protects hiECs from killing by both NK cells and macrophages.

[0388] Example 2 Macrophage phagocytosis was also measured using flow cytometry. Human B2M transduced with or without a lentiviral vector expressing CD24 (CD24tg) was used. - / - CIITA - / - iPSCs, Essential 8 Fl Cells were cultured on 10 cm dishes coated with diluted feeder-free MATRIGEL (hESC Establishment, BD Biosciences, San Jose, CA) in exo culture medium (Thermo Fisher Scientific). Cells were harvested at 60% confluence and fluorescently labeled with calcein AM (Invitrogen) according to the manufacturer's instructions by suspending the cells in PBS + 1:30,000 calcein AM at 37°C for 15 min and washing twice with 40 ml of PBS before co-culture. Cells were then co-cultured at an effector-to-target cell (E:T) ratio of 1:2 with human macrophages stimulated with 50 ng / ml human TGFβ1 and 50 ng / ml human IL-10 for 4 days. After co-culture, the plates are placed on ice, centrifuged at 400 g for 5 minutes at 4°C, and the phagocytosis assay is terminated by staining with A647-labeled anti-CD11b (Clone M1 / 70, BioLegend) to identify human macrophages. The assay is analyzed by flow cytometry on an Attune NxT flow analyzer. Phagocytosis is measured as the number of CD11b+calcneurin+ macrophages and quantified as a percentage of total CD11b+ macrophages. B2M - / - CIITA - / - hiECs (without CD24) are significantly phagocytosed, but B2M - / - CIITA - / - CD24 tg hiECs are protected from phagocytosis. Blockade with 10 μg / ml of anti-CD24 antibody (Clone SN3, Novus Biologics) eliminates the protective effect. Macrophage phagocytosis is also measured using flow cytometry. Human B2M cells transduced with or without a lentiviral vector expressing CD24 (CD24tg) were transduced with or without a lentiviral vector expressing CD24. - / - CIITA - / -iPSCs were cultured on 10 cm dishes coated with diluted feeder-free MATRIGEL (hESC Establishment, BD Biosciences, San Jose, CA) in Essential 8 Flex medium (Thermo Fisher Scientific). Cells were harvested at 60% confluence and fluorescently labeled with calcein AM (Invitrogen) according to the manufacturer's instructions by suspending the cells in PBS + 1:30,000 calcein AM at 37°C for 15 min and washing twice with 40 ml of PBS before co-culture. Cells were then co-cultured at an effector-to-target cell (E:T) ratio of 1:2 with human macrophages stimulated with 50 ng / ml human TGFβ1 and 50 ng / ml human IL-10 for 4 days. After co-culture, the plates are placed on ice, centrifuged at 400 g for 5 minutes at 4°C, and the phagocytosis assay is terminated by staining with A647-labeled anti-CD11b (Clone M1 / 70, BioLegend) to identify human macrophages. The assay is analyzed by flow cytometry on an Attune NxT flow analyzer. Phagocytosis is measured as the number of CD11b+calcneurin+ macrophages and quantified as a percentage of total CD11b+ macrophages. B2M - / - CIITA - / - hiECs (without CD24) are significantly phagocytosed, but B2M - / - CIITA - / - CD24 tg hiECs are protected from phagocytosis. Blockade with 10 μg / ml of anti-CD24 antibody (Clone SN3, Novus Biologics) eliminates the protective effect.

[0389] All heading and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, one of ordinary skill in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0390] All references cited in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0391] Many modifications and variations of this application may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The examples are provided by way of example only, and this application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. An isolated cell, comprising reduced expression of MHC class I and / or MHC class II human leukocyte antigens, and a modification to increase expression of CD24 on said cell.

2. The isolated cell of claim 1 , wherein the cell comprises reduced expression of MHC class I and MHC class II human leukocyte antigens.

3. 3. The isolated cell of claim 1 or 2, wherein the cell further comprises a genetic modification that targets the CIITA gene with a rare-cutting endonuclease that selectively inactivates the CIITA gene.

4. 4. The isolated cell of any one of claims 1 to 3, wherein the cell further comprises a modification to increase expression in the cell of a polypeptide selected from the group consisting of CD47, DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, CCL21, Mfge8, and Serpinb9.

5. The isolated cell of claim 4, wherein the cell further comprises a modification to increase expression of CD47 in the cell.

6. 6. The isolated cell of any one of claims 1 to 5, wherein the cell further comprises a genetic modification that targets the B2M gene with a rare-cutting endonuclease that selectively inactivates the B2M gene.

7. 7. The isolated cell of any one of claims 1 to 6, wherein the cell further comprises a genetic modification that targets the NLRC5 gene with a rare-cutting endonuclease that selectively inactivates the NLRC5 gene.

8. 8. The isolated cell of claim 3, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

9. The isolated cell of any one of claims 3 to 8, wherein the genetic modification targeting the CIITA gene with 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 CIITA gene.

10. The isolated cell of any one of claims 6 to 9, wherein the genetic modification targeting the B2M gene with 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 B2M gene.

11. The isolated cell of any one of claims 7 to 10, wherein the genetic modification targeting the NLRC5 gene with 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.

12. The isolated cell of any one of claims 1 to 11, wherein the modification to increase CD24 expression comprises introducing into the cell an expression vector comprising a polynucleotide sequence encoding CD24.

13. 13. The isolated cell of claim 12, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31.

14. 14. The isolated cell of claim 12 or 13, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 28 to 31.

15. 15. The isolated cell of any one of claims 4 to 14, wherein the modification to increase expression of one or more polypeptides selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 comprises introducing into the cell an expression vector comprising one or more polynucleotide sequences encoding one or more polypeptides selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35.

16. The isolated cell of any one of claims 4 to 15, wherein the modification to increase CD47 expression comprises introducing into the cell an expression vector comprising a polynucleotide sequence encoding CD47.

17. The isolated cell of any one of claims 14 to 16, wherein the expression vector is an inducible expression vector.

18. The isolated cell of any one of claims 14 to 17, wherein the expression vector is a viral vector.

19. 19. The isolated cell of any one of claims 1 to 18, wherein the modification to increase expression of CD24 comprises introducing a polynucleotide sequence encoding CD24 into a selected locus of the cell.

20. 20. The isolated cell of claim 19, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31.

21. 21. The isolated cell of claim 19 or 20, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 28 to 31.

22. 22. The isolated cell of any one of claims 4-15 or 19-21, wherein the modification to increase expression of one or more polypeptides selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 comprises introducing into a selected locus of the cell a polynucleotide sequence encoding one or more polypeptides selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35.

23. 23. The isolated cell of claim 22, wherein the modification to increase expression of CD47 comprises introducing a polynucleotide sequence encoding CD47 into a selected locus of the cell.

24. 24. The isolated cell of any one of claims 19 to 23, wherein the selected locus for the polynucleotide sequence encoding CD24 and / or the selected locus for the polynucleotide sequence encoding one selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35 is a safe harbor locus.

25. 25. The isolated cell of claim 24, wherein the safe harbor is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

26. 26. The isolated cell of any one of claims 1 to 25, further comprising an inducible suicide switch.

27. 27. The isolated cell of any one of claims 1 to 26, wherein the cell is selected from the group consisting of stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

28. A method for preparing cells containing CD24, the method comprising introducing into the cells an expression vector comprising a polynucleotide sequence encoding CD24, thereby producing the cells containing CD24.

29. 29. The method of claim 28, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31.

30. 30. The method of claim 28 or 29, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 28 to 31.

31. 31. The method of any one of claims 28 to 30, wherein the cells comprising CD24 further comprise a genetic modification that targets the CIITA gene, the genetic modification comprising a rare-cutting endonuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease for targeting the CIITA gene.

32. 32. The method of claim 31, wherein the genetic modification comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting the CIITA gene.

33. The method of any one of claims 28 to 32, wherein the expression vector is an inducible expression vector.

34. The method of any one of claims 28 to 33, wherein the expression vector is a viral vector.

35. 35. The method of any one of claims 28-34, wherein the cells comprising CD24 further comprise a second expression vector comprising a polynucleotide sequence encoding one selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35.

36. The method of any one of claims 28 to 35, wherein the second expression vector comprises a polynucleotide sequence encoding CD47.

37. 37. The method of claim 35 or 36, wherein the second expression vector is an inducible expression vector.

38. The method of any one of claims 35 to 37, wherein the second expression vector is a viral vector.

39. 39. The method of any one of claims 28 to 38, wherein the cells comprising CD24 further comprise a genetic modification that targets the B2M gene, comprising a rare-cutting endonuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease, for specifically targeting the B2M gene.

40. 40. The method of claim 39, wherein the genetic modification comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting the B2M gene.

41. The method of any one of claims 28 to 40, wherein the cells comprising CD24 further comprise a genetic modification that targets the NLRC5 gene, comprising a rare-cutting endonuclease selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease, for specifically targeting the NLRC5 gene.

42. 42. The method of claim 41, wherein the genetic modification comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting the NLRC5 gene.

43. 43. The method of any one of claims 28 to 42, wherein the cells are selected from the group consisting of stem cells, differentiated cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

44. 1. A method for preparing hypoimmunogenic stem cells, comprising introducing a polynucleotide sequence encoding CD24 into a selected locus of said stem cells, thereby producing hypoimmunogenic stem cells.

45. 45. The method of claim 44, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 28-31.

46. The method of claim 44 or 45, wherein the polynucleotide sequence encoding CD24 is a nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 28 to 31.

47. The method of any one of claims 44 to 46, further comprising generating a genetic modification targeting the CIITA gene in the stem cell, comprising introducing into the stem cell a rare-cutting endonuclease that selectively inactivates the CIITA gene, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

48. 48. The method of claim 47, wherein said introducing said rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting said CIITA gene.

49. 49. The method of any one of claims 44 to 48, wherein the selected locus for the polynucleotide sequence encoding CD24 is a safe harbor locus.

50. 50. The method of claim 49, wherein the safe harbor locus for the polynucleotide sequence encoding CD24 is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

51. 51. The method of any one of claims 44 to 50, further comprising introducing into the selected locus of the stem cell a polynucleotide sequence encoding a polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35.

52. 52. The method of any one of claims 44 to 51, further comprising introducing a polynucleotide sequence encoding CD47 into the selected locus of said stem cells.

53. 53. The method of claim 51 or 52, wherein the selected loci are safe harbor loci.

54. 54. The method of claim 53, wherein the safe harbor locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, the CLYBL locus, the ROSA26 locus, and the SHS231 locus.

55. 55. The method of any one of claims 44 to 54, further comprising generating a genetic modification in the stem cell that targets the B2M gene, comprising introducing into the stem cell a rare-cutting endonuclease that selectively inactivates the B2M gene, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

56. 56. The method of Claim 55, wherein said introducing said rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting the B2M gene.

57. A genetic modification targeting the NLRC5 gene in stem cells, comprising introducing into the stem cells a rare-cutting endonuclease that selectively inactivates the NLRC5 gene.

57. The method of any one of claims 44 to 56, further comprising generating a rare-cutting endonuclease, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

58. 58. The method of claim 57, wherein said introducing said rare-cutting endonuclease comprises introducing a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid for specifically targeting the NLRC5 gene.

59. 59. The method of any one of claims 44 to 58, further comprising introducing into said stem cells an expression vector comprising an inducible suicide switch.

60. A method for preparing differentiated hypoimmunogenic cells, the method comprising culturing the hypoimmunogenic stem cells prepared according to the method of any one of claims 44 to 59 under differentiation conditions, thereby preparing differentiated hypoimmunogenic cells.

61. 61. The method of claim 60, wherein the differentiation conditions are suitable for differentiation of stem cells into a cell type selected from the group consisting of cardiac cells, neural cells, endothelial cells, T cells, pancreatic islet cells, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

62. 62. A method of treating a patient in need of cell therapy, comprising administering a population of differentiated, hypoimmunogenic cells prepared according to the method of claim 60 or 61.

63. Cells that express CD24 and have reduced expression of MHC class I human leukocyte antigens.

64. Cells that express CD24 and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

65. Cells that do not express CIITA, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

66. Cells that do not express B2M, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

67. Cells that do not express NLRC5, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

68. A cell that expresses CD24 and at least one polypeptide selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and has reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

69. Cells that express CD24 and CD47 and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

70. does not express CIITA, and expresses CD24 and at least one polypeptide selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35; Cells having reduced expression of one or more MHC class I and / or MHC class II human leukocyte antigens.

71. Cells that do not express CIITA, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

72. Cells that do not express CIITA and B2M, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

73. A cell that does not express CIITA and B2M, but expresses CD24 and at least one polypeptide selected from the group consisting of CD47, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and has reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

74. Cells that do not express CIITA and B2M, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

75. Cells that do not express CIITA and NLRC5, express CD24, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

76. A cell that does not express CIITA and NLRC5, but expresses CD24 and at least one polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and has reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

77. Cells that do not express CIITA and NLRC5, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

78. A cell that does not express CIITA, B2M, and NLRC5, but expresses CD24 and at least one polypeptide selected from the group consisting of CD47, CD35, DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, C1 inhibitor, CD46, CD55, CD59, and IL-35, and has reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

79. Cells that do not express CIITA, B2M, and NLRC5, express CD24 and CD47, and have reduced expression of MHC class I and / or MHC class II human leukocyte antigens.

80. 80. The cell of any one of claims 63 to 79, wherein the cell is selected from the group consisting of stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

81. 81. A differentiated cell produced from the pluripotent stem cell or induced pluripotent stem cell of claim 80 by culturing under differentiation conditions that produce differentiated cells selected from the group consisting of cardiac cells, neural cells, endothelial cells, T cells, pancreatic islet cells, retinal pigment epithelial (RPE) cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

82. An isolated stem cell comprising an exogenous CD24 polypeptide.

83. 83. The isolated cell of claim 82, wherein the cell expresses a nucleotide sequence encoding a CD24 polypeptide having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:

31.

84. 84. The isolated cell of claim 82 or 83, wherein the CD24 polypeptide is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:

31.

85. 85. The isolated cell of any one of claims 82 to 84, wherein the cell has reduced expression of MHC class I human leukocyte antigens.

86. 86. The isolated cell of any one of claims 82 to 85, wherein the cell has reduced expression of MHC II human leukocyte antigen.

87. 87. The isolated cell of any one of claims 82 to 86, wherein the cell has reduced expression of MHC class I and MHC II human leukocyte antigens.

88. 88. The isolated cell of any one of claims 82 to 87, wherein the cell has reduced expression of CIITA.

89. 89. The isolated cell of any one of claims 82 to 88, wherein the cell has reduced expression of B2M.

90. 90. The isolated cell of any one of claims 82 to 89, wherein the cell has reduced expression of NLRC5.

91. 91. The isolated cell of any one of claims 82 to 90, further comprising a genomic modification that targets CIITA to reduce expression of CIITA.

92. 92. The isolated cell of any one of claims 82 to 91, further comprising a genomic modification that targets B2M to reduce expression of B2M.

93. 93. The isolated cell of any one of claims 82 to 92, further comprising a genomic modification that targets NLRC5 to reduce expression of NLRC5.

94. 94. The isolated cell of any one of claims 91 to 93, wherein the genomic modification comprises a rare-cutting endonuclease.

95. 95. The isolated cell of claim 94, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

96. 96. The isolated cell of claim 94 or 95, wherein the rare-cutting endonuclease comprises a polynucleotide encoding a Cas protein or a Cas protein that targets CIITA.

97. 97. The isolated cell of claim 96, further comprising at least one guide ribonucleic acid sequence recognized by the Cas protein that targets CIITA.

98. The isolated cell of claim 97, wherein the at least one guide ribonucleic acid sequence for targeting CIITA is selected from the group consisting of SEQ ID NOs: 5184 to 36352 of WO2016 / 183041.

99. 96. The isolated cell of claim 94 or 95, wherein the rare-cutting endonuclease comprises a polynucleotide encoding a Cas protein or a Cas protein that targets B2M.

100. 100. The isolated cell of Claim 99, further comprising at least one guide ribonucleic acid sequence recognized by said Cas protein that targets B2M.

101. 101. The isolated cell of claim 100, wherein the at least one guide ribonucleic acid sequence for targeting B2M is selected from the group consisting of SEQ ID NOs: 81240 to 85644 of WO2016 / 183041.

102. 96. The isolated cell of claim 94 or 95, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein that targets NLRC5.

103. The isolated cell of claim 102, further comprising at least one guide ribonucleic acid sequence recognized by the Cas protein that targets NLRC5.

104. The isolated cell of claim 103, wherein the at least one guide ribonucleic acid sequence for targeting NLRC5 is selected from the group consisting of SEQ ID NOs: 36353 to 81239 of WO2016 / 183041.

105. 105. The isolated cell of any one of claims 82 to 104, further comprising a gene expression modification to reduce expression of CIITA.

106. 106. The isolated cell of any one of claims 82 to 105, further comprising a gene expression modification to reduce expression of B2M.

107. 107. The isolated cell of any one of claims 82 to 106, further comprising a gene expression modification to reduce expression of NLRC5.

108. 108. The isolated cell of any one of claims 105-107, wherein the gene expression modification comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule.

109. 109. The isolated cell of any one of claims 82-108, further comprising an exogenous immune modulator selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35.

110. 110. The isolated cell of any one of claims 82-109, further comprising one or more exogenous immune modulators selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35.

111. 111. The isolated cell of any one of claims 82-110, wherein the cell is selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

112. An isolated cell produced from the stem cell of any one of claims 82 to 111 under differentiation conditions.

113. The isolated cell of any one of claims 82 to 112, wherein the cell is hypoimmunogenic.

114. 1. A method for preparing stem cells containing an exogenous CD24 polypeptide, the method comprising introducing an expression vector comprising a nucleotide sequence encoding a CD24 polypeptide having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:

31.

115. 115. The method of claim 114, wherein the CD24 polypeptide is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:

31.

116. 116. The method of claim 114 or 115, wherein the expression vector is an inducible expression vector.

117. The method of any one of claims 114 to 116, wherein the expression vector is a viral vector.

118. 118. The method of any one of claims 114 to 117, wherein the expression vector specifically targets a safe harbor locus.

119. 119. The method of claim 118, wherein the safe harbor locus is the AAVS1 locus.

120. 120. The method of any one of claims 114 to 119, further comprising introducing into the cell a rare-cutting endonuclease that selectively inactivates the CIITA gene.

121. 121. The method of any one of claims 114 to 120, further comprising introducing into the cell a rare-cutting endonuclease that selectively inactivates the B2M gene.

122. The method of any one of claims 114 to 121, further comprising introducing into the cell a rare-cutting endonuclease that selectively inactivates the NLRC5 gene.

123. 123. The method of any one of claims 120 to 122, wherein the rare-cutting endonuclease is selected from the group consisting of a Cas protein, a TALE nuclease, a zinc finger nuclease, a meganuclease, and a homing nuclease.

124. The method of claim 120 or 123, further comprising introducing at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein.

125. The method of claim 124, wherein the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOs: 5184 to 36352 of WO2016 / 183041.

126. The method further comprises introducing at least one guide ribonucleic acid sequence for specifically targeting the B2M gene, wherein the rare-cutting endonuclease is a Cas protein or 124. The method of claim 121 or 123, comprising a polynucleotide encoding a Cas protein.

127. 127. The method of claim 126, wherein 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 to 85644 of WO2016 / 183041.

128. The method of claim 122 or 123, further comprising introducing at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene, wherein the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein.

129. The method of claim 128, wherein 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 to 81239 of WO2016 / 183041.

130. 130. The method of any one of claims 114-129, further comprising introducing a gene expression modifying molecule into the cell to reduce expression of CIITA, wherein the gene expression modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets CIITA.

131. 131. The method of any one of claims 114-130, further comprising introducing a gene expression modifying molecule into the cell to reduce expression of B2M, wherein the gene expression modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets B2M.

132. The method of any one of claims 114 to 131, further comprising introducing a gene expression modifying molecule into the cell to reduce expression of NLRC5, wherein the gene expression modifying molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule that specifically targets NLRC5.

133. 133. The method of any one of claims 114 to 132, further comprising introducing an expression vector comprising a nucleotide sequence encoding a tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35.

134. 134. The method of any one of claims 114-133, further comprising introducing at least two expression vectors, wherein the first expression vector comprises a first nucleotide sequence encoding a first tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35, and the second expression vector comprises a second nucleotide sequence encoding a different tolerogenic polypeptide selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35.

135. 135. The method of claim 133 or 134, wherein the expression vector, the first expression vector, and / or the second expression vector is an inducible expression vector.

136. 136. The method of any one of claims 133 to 135, wherein the expression vector, the first expression vector, and / or the second expression vector is a viral vector.

137. 137. The method of any one of claims 133-136, wherein the expression vector, the first expression vector, and / or the second expression vector specifically targets a safe harbor locus.

138. 138. The method of claim 137, wherein the safe harbor locus is the AAVS1 locus.

139. 139. The method of any one of claims 114 to 138, further comprising introducing into said stem cells an expression vector comprising an inducible suicide switch.

140. 140. The method of any one of claims 114 to 139, wherein the stem cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, and adult stem cells.

141. 141. The method of any one of claims 114 to 140, wherein the stem cells have reduced expression of MHC class I human leukocyte antigens compared to unmodified stem cells.

142. 142. The method of any one of claims 114 to 141, wherein the stem cells have reduced expression of MHC class II human leukocyte antigens compared to unmodified stem cells.

143. 143. The method of any one of claims 114 to 142, wherein the stem cells have reduced expression of MHC class I and class II human leukocyte antigens compared to unmodified stem cells.

144. The method of any one of claims 114 to 143, wherein the stem cells are hypoimmunogenic.

145. A method for preparing differentiated cells, comprising culturing stem cells prepared according to the method of any one of claims 114 to 144 under differentiation conditions, thereby preparing differentiated cells.

146. 146. The method of claim 145, wherein the differentiation conditions are suitable for differentiating stem cells into a cell type selected from the group consisting of cardiac cells, hepatic cells, kidney cells, pancreatic cells, neural cells, immune cells, mesenchymal cells, and endothelial cells.

147. 147. A method of treating a patient in need of cell therapy, comprising administering a population of differentiated cells prepared according to the method of claim 145 or 146.

148. Stem cells expressing exogenous CD24 polypeptide and reduced expression levels of MHC class I human leukocyte antigen.

149. Stem cells expressing exogenous CD24 polypeptide and reduced expression levels of MHC class II human leukocyte antigen.

150. Stem cells that express exogenous CD24 polypeptide and reduced expression levels of MHC class I and class II human leukocyte antigens.

151. Stem cells expressing exogenous CD24 polypeptide and reduced expression levels of CIITA.

152. Stem cells expressing exogenous CD24 polypeptide and reduced expression levels of B2M.

153. Stem cells expressing exogenous CD24 polypeptide and reduced expression levels of NLRC5.

154. Stem cells that express exogenous CD24 polypeptide and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

155. A stem cell expressing an exogenous CD24 polypeptide and one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35.

156. A stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of CIITA.

157. A stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of B2M.

158. A stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of NLRC5.

159. A stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

160. A differentiated cell generated from a stem cell that expresses exogenous CD24 polypeptide and reduced expression levels of MHC class I human leukocyte antigen.

161. A differentiated cell generated from a stem cell that expresses exogenous CD24 polypeptide and reduced expression levels of MHC class II human leukocyte antigen.

162. A differentiated cell generated from a stem cell that expresses exogenous CD24 polypeptide and reduced expression levels of MHC class I and class II human leukocyte antigens.

163. A differentiated cell generated from a stem cell expressing an exogenous CD24 polypeptide and a reduced expression level of CIITA.

164. A differentiated cell generated from a stem cell that expresses an exogenous CD24 polypeptide and a reduced expression level of B2M.

165. A differentiated cell generated from a stem cell expressing exogenous CD24 polypeptide and reduced expression levels of NLRC5.

166. Differentiated cells generated from stem cells that express exogenous CD24 polypeptide and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

167. Exogenous CD24 polypeptide and HLA-C, HLA-E, HLA-G, PD-L1, A differentiated cell generated from a stem cell that expresses one or more immune tolerogenic factors selected from the group consisting of CTLA-4-Ig, C1 inhibitor, and IL-35.

168. A differentiated cell generated from a stem cell that expresses an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of CIITA.

169. A differentiated cell generated from a stem cell that expresses an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of B2M.

170. A differentiated cell generated from a stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and a reduced expression level of NLRC5.

171. A differentiated cell generated from a stem cell expressing an exogenous CD24 polypeptide, one or more tolerogenic factors selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, C1 inhibitor, and IL-35, and reduced expression levels of CIITA, B2M, NLRC5, and combinations thereof.

172. The stem cell of any one of claims 148 to 159, wherein the cell is low immunogenic.

173. The differentiated cell of any one of claims 160 to 172, wherein the cell is low immunogenic.

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