Safety switches for regulating gene expression

Abstract

This invention provides a method for controlling the immunogenicity of manipulated cells. [Solution] The method is, (a) Obtaining isolated cells, (b) To produce engineered cells by introducing into the isolated cells (i) a nucleic acid containing an inducible RNA polymerase promoter operably linked to an shRNA sequence targeting an immunosuppressive factor, and (ii) a nucleic acid containing a promoter operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter, (c) Exposing the manipulated cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the manipulated cells, Includes.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Applications No. 62 / 962,730, No. 62 / 962,739, and No. 62 / 962,764, filed on 17 January 2020, under Section 119(e) of the U.S. Patent Act, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listings This application has been filed electronically, along with a sequence listing. This sequence listing is provided as a file titled SANA006WO1SeqList.txt, created on January 14, 2021, and measuring 33,948 bytes. The electronic information of this sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Degenerative diseases pose a disproportionate threat to human health. Often age-related, these diseases lead to the progressive decline of affected tissues and organs, ultimately resulting in physical disability and death in the affected individual. The potential of regenerative medicine lies in replacing diseased or missing cells with new, healthy cells. In the last five years, a new paradigm for regenerative medicine has emerged: the use of human pluripotent stem cells (hPSCs) to generate any adult cell type for transplantation into patients. In principle, hPSC-based cell therapies have the potential to treat most, if not all, degenerative diseases, but the success of such therapies can be limited by the immune response of the individual.

[0004] Strategies considered to overcome immune rejection include HLA matching (e.g., identical twin or umbilical cord banking), administration of immunosuppressants to the subject, blocking antibodies, myelosuppression / mixed chimerism, repertoire of HLA-matched stem cells, and autologous stem cell therapy.

[0005] There remains a need for novel methods, compositions, and techniques to overcome immune rejection associated with cell therapy. [Overview of the project]

[0006] In one embodiment, a method for controlling the immunogenicity of engineered cells is provided herein, the method comprising (a) obtaining isolated cells; (b) introducing into the isolated cells a nucleic acid comprising (i) an inducible RNA polymerase promoter operably linked to an shRNA sequence targeting an immunosuppressive factor, and (ii) a nucleic acid comprising a promoter operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter, thereby producing engineered cells; and (c) exposing the engineered cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the cells.

[0007] In one embodiment, a method for controlling the immunogenicity of engineered cells is provided herein, the method comprising: (a) obtaining isolated cells; (b) introducing nucleic acids into the isolated cells, comprising (i) a sequence encoding an inducible degron element operably linked to an immunosuppressive factor, or (ii) a sequence encoding an immunosuppressive factor operably linked to an inducible degron element, to produce engineered cells; and (c) exposing the engineered cells to an exogenous factor for activating the inducible degron element, thereby engineering the cells. This includes controlling the immunogenicity of cells.

[0008] In another embodiment, a method for controlling the immunogenicity of engineered cells is provided herein, the method comprising (a) obtaining isolated cells; (b) introducing into the isolated cells a first construct comprising (i) a first promoter and an immunosuppressant gene from the 5' to 3' end; (ii) a second construct comprising a second promoter and a nucleic acid sequence encoding Cas9 or a variant thereof from the 5' to 3' end; and (ii) a transactivator element corresponding to an inducible RNA polymerase promoter, an immunosuppressant-targeting guide RNA (gRNA) sequence, a third promoter, and an inducible RNA polymerase promoter from the 5' to 3' end; and (c) exposing the engineered cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the engineered cells.

[0009] In another embodiment, a method for controlling the immunogenicity of engineered cells is provided herein, the method comprising (a) obtaining isolated cells; (b) introducing into the isolated cells a nucleic acid comprising (i) an inducible RNA polymerase promoter operably linked to an immune signaling factor gene, and (ii) a nucleic acid comprising a promoter operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter, thereby producing engineered cells; and (c) exposing the engineered cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the engineered cells.

[0010] In some embodiments, the method further includes administering the manipulated cells to the target before step (c).

[0011] In some embodiments, step (b) of any of these methods comprises introducing into the isolated cells a single nucleic acid construct comprising (i) an inducible RNA polymerase promoter operably linked to an shRNA sequence targeting an immunosuppressive factor, and (ii) a promoter operably linked to a trans-activating factor element. In some embodiments, the construct comprises, from the 5' end to the 3' end, an inducible RNA polymerase promoter, an shRNA sequence, a promoter, and a trans-activating factor element.

[0012] In some embodiments, step (b) comprises introducing into the isolated cells a single nucleic acid construct comprising (i) an inducible RNA polymerase promoter operably linked to an immune signaling factor gene, and (ii) a promoter operably linked to a trans-activating factor element.

[0013] In some embodiments, the construct comprises, from the 5' end to the 3' end, an inducible RNA polymerase promoter, an immune signaling factor gene, a promoter, and a trans-activating factor element.

[0014] In some embodiments, the isolated cells are engineered to express an immunosuppressive factor exogenously. In some embodiments, the isolated cells overexpress the immunosuppressive factor in the absence of an exogenous factor that activates the trans-activating factor element.

[0015] In some embodiments, the inducible RNA polymerase promoter is the U6Tet promoter. In some embodiments, the inducible RNA polymerase promoter is the U6Tet promoter, the trans-activating factor element is the Tet repressor element, and the exogenous factor is tetracycline or a derivative thereof. In some embodiments, the inducible RNA polymerase promoter is the TRE promoter, the trans- activating factor element is the Tet-On element, and the exogenous factor is tetracycline or a derivative thereof.

[0016] In some embodiments, a flexible linker connects the inducible degron element to the immunosuppressive factor. In some embodiments, the flexible linker is selected from the group consisting of (GSG)n, (GGGS)n, and (GGGSGGGS)n, where n is from 1 to 10 in the sequence.

[0017] In some embodiments, step (b) includes introducing into the isolated cell a single nucleic acid construct comprising a promoter operably linked to the nucleic acid.

[0018] In some embodiments, the promoter is a constitutive promoter selected from the group consisting of the EF1A promoter, the EFS promoter, the CMV promoter, the CAGGS promoter, the SV40 promoter, the COPIA promoter, the ACT5C promoter, the TRE promoter, the CBh promoter, the PGK promoter, and the UBC promoter. In some embodiments, the first promoter, the second promoter, and / or the third promoter are each independently a constitutive promoter selected from the group consisting of the EF1A promoter, the EFS promoter, the CMV promoter, the CAGGS promoter, the SV40 promoter, the COPIA promoter, the ACT5C promoter, the TRE promoter, the CBh promoter, the PGK promoter, and the UBC promoter.

[0019] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0020] In some embodiments, the construct comprises, from the 5' end to the 3' end, a U6Tet promoter, an shRNA sequence targeting CD47, an EF1a promoter, and a Tet repressor element, and the exogenous factor is tetracycline or a derivative thereof.

[0021] In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0022] In some embodiments, the inducible degron element is selected from the group consisting of ligand-inducible degron elements, peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements. In some embodiments, the ligand-inducible degron element is selected from small molecule-assisted shutoff (SMASH) degron elements, Shield-1 responsive degron elements, auxin-responsive degron elements, and rapamycin-responsive degron elements. In some embodiments, the ligand-inducible degron element is a small molecule-assisted shutoff (SMASH) degron element, and the exogenous factor is asunaprevir.

[0023] In some embodiments, the construct further includes 5' homology arms and 3' homology arms for target incorporation to safe harbor loci selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5 loci.

[0024] In some embodiments, the isolated cells are isolated human cells further comprising the deletion or reduction of expression of MHC class I human leukocyte antigen and / or the deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells. The isolated human cells further include deletions or reduced expression of CIITA, B2M, and / or NLRC5.

[0025] In some embodiments, the isolated human cells are low immunogenic and are either stem cells or differentiated cells thereof, wherein the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells, and the differentiated cells are selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells. In some embodiments, the isolated human cells are low immunogenic. In some embodiments, the isolated human cells are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0026] In another embodiment, a construct is provided herein that comprises, from its 5' end to its 3' end, an inducible RNA polymerase promoter, an immunosuppressive factor-targeting shRNA sequence, a constitutive promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter.

[0027] In one embodiment, a construct is provided herein that comprises, from its 5' end to its 3' end, an inducible RNA polymerase promoter, an immune signaling factor gene, a promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter.

[0028] In some embodiments, the inducible RNA polymerase promoter is the U6Tet promoter. In many embodiments, the inducible RNA polymerase promoter is the TRE promoter.

[0029] In some embodiments, the immunosuppressant of the construct is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the immune signaling factor of the construct is selected from the group consisting of B2M, MIC-A, MIC-B, HLA-A, HLA-B, HLA-C, RFXANK, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D.

[0030] In some embodiments, the promoter of the construct is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0031] In some embodiments, the construct includes a U6Tet promoter, a CD47-targeting shRNA sequence, an EF1a promoter, and a Tet repressor element from the 5' end to the 3' end.

[0032] In some embodiments, the construct includes a TRE promoter, an immune signaling factor gene, an EF1a promoter, and a Tet-On element from the 5' end to the 3' end.

[0033] In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0034] Furthermore, compositions are also provided that include isolated cells containing any of the constructs described herein.

[0035] Furthermore, compositions are provided comprising isolated cells containing any of the constructs described herein, wherein the isolated cells are engineered to exogenously express an immunosuppressive factor. In some embodiments, the isolated cells overexpress an immunosuppressive factor in the absence of an exogenous factor that activates a transactivator element. In some embodiments, the isolated cells are exposed to an exogenous factor for activating the transactivator element. In some embodiments, the isolated cells are stem cells selected from the group consisting of embryonic stem cells, pluripotent stem cells, and adult stem cells.

[0036] In some embodiments, the composition comprises isolated differentiated cells prepared by culturing one of the stem cells outlined herein under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0037] In some embodiments, methods for treating patients requiring cell therapy are provided herein, the methods comprising (a) administering to a patient any of the compositions outlined herein, and (b) exposing the composition to an exogenous factor for activating an inducible RNA polymerase promoter, thereby controlling the cellular immunogenicity of the composition.

[0038] (i) reduction or silencing of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) a factor selected from the group consisting of inducible shRNA targeting CD47, inducible degron elements that regulate CD47, or SMASH degron elements that regulate CD47.

[0039] Also provided herein are pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) a factor selected from the group consisting of inducible shRNA targeting CD47, inducible degron elements that regulate CD47, or SMASH degron elements that regulate CD47.

[0040] In addition, pluripotent stem cells comprising (i) reduction or silencing of MHC class I and / or MHC class II molecules, (ii) overexpression of CD47, (iii) Cas9 or its variants, and (iv) inducible guide RNA targeting CD47 are outlined herein.

[0041] Furthermore, pluripotent stem cells comprising (i) reduced or silencing of B2M and CIITA expression, (ii) overexpression of CD47, (iii) Cas9 or its variants, and (iv) inducible guide RNA targeting CD47 are outlined herein.

[0042] Furthermore, pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an inducible proteolytic system for regulating CD47 expression, selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolytic chimeric molecules, and antibodies for targeted degradation are outlined herein.

[0043] Furthermore, (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) small molecule co-blocking (SMASH) system, seal Pluripotent stem cells comprising an inducible proteolytic system for regulating CD47 expression, selected from the group consisting of do-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolytic chimeric molecules, and antibodies for targeted degradation, are outlined herein.

[0044] Pluripotent stem cells are provided herein that include (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an RNA regulatory system for regulating CD47 expression, selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

[0045] Pluripotent stem cells are provided herein that include (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) an RNA regulatory system for regulating CD47 expression, selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

[0046] Pluripotent stem cells are provided herein that include (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) a DNA regulatory system for regulating CD47 expression, selected from the group consisting of tissue-specific promoter expression systems, inducible promoter expression systems, molecularly controlled riboswitch systems, and inducible nuclease-based genome editing systems.

[0047] Pluripotent stem cells are provided herein that include (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) a DNA regulatory system for regulating CD47 expression, selected from the group consisting of tissue-specific promoter expression systems, inducible promoter expression systems, molecularly controlled riboswitch systems, and inducible nuclease-based genome editing systems.

[0048] Pluripotent stem cells are provided herein that include (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an inducible system for modulating the expression of CD47. In some embodiments, the inducible system reduces or diminishes the expression of CD47 in the cells.

[0049] Also provided herein are pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) an inducible system for regulating CD47 expression. In some embodiments, the inducible system reduces or diminishes CD47 expression.

[0050] In some cases, differentiated cells derived from one of the outlined pluripotent stem cells are provided, selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0051] In one embodiment, a construct is outlined that includes a promoter, an inducible degron element, an optional sequence encoding a flexible linker, and an immunosuppressive factor gene, from the 5' end to the 3' end.

[0052] In another embodiment, a construct comprising a promoter, an immunosuppressive factor gene, an optional sequence encoding a flexible linker, and an inducible degron element from the 5' end to the 3' end. This will be outlined.

[0053] In some embodiments, the promoter is a constitutive promoter selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0054] In many embodiments, the flexible linker is (GSG) n (Sequence ID 3), (GGGS) n (Sequence ID 1), and (GGGSGGGS) n Selected from the group consisting of (Sequence ID 2), where n is between 1 and 10.

[0055] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0056] In some embodiments, the inducible degron element is selected from the group consisting of ligand-inducible degron elements, inducible peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements. In some embodiments, the ligand-inducible degron element is selected from small molecule co-blocking (SMASH) degron elements, shield-1 responsive degron elements, auxin-responsive degron elements, and rapamycin-responsive degron elements.

[0057] In some embodiments, the construct further includes 5' homology arms and 3' homology arms for target integration to intragenomic safe harbor loci selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5.

[0058] A composition is provided, comprising isolated cells containing one of the constructs described.

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

[0060] A composition is provided which comprises isolated differentiated cells prepared by culturing one of the described stem cells under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0061] In some embodiments, methods for treating patients requiring cell therapy are provided herein, the methods comprising (a) administering a composition described herein to a patient, and (b) exposing the composition to an exogenous factor for activating an inducible degron element, thereby controlling the cellular immunogenicity of the composition.

[0062] In some embodiments, a composition is provided herein comprising isolated cells containing a DNA-targeted nuclease system for controlling the immunogenicity of cells, the composition comprising: (a) a first element comprising a first promoter and an immunosuppressive factor gene from the 5' end to the 3' end; (b) a second element comprising a second promoter and a nucleic acid sequence encoding Cas9 or a variant thereof from the 5' end to the 3' end; and (c) from the 5' end to the 3' end The third element comprises an inducible RNA polymerase promoter, a guide RNA (gRNA) sequence targeting an immunosuppressive factor, a third promoter, and a transactivator element corresponding to the inducible promoter. In certain embodiments, the immunogenicity of the cell is controllable when the cell is exposed to an exogenous factor to induce the activity of the transactivator element. In some embodiments, the inducible RNA polymerase promoter is a U6Tet promoter, the transactivator element is a Tet repressor element, and the exogenous factor is tetracycline or a derivative thereof.

[0063] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0064] In some embodiments, the first promoter, the second promoter, and / or the third promoter are constitutive promoters independently selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0065] In some embodiments, the isolated human cells further include deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated human cells further include deletion or reduced expression of CIITA, B2M, and / or NLRC5.

[0066] In some embodiments, isolated human cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0067] The following compositions are provided herein, comprising isolated differentiated cells prepared by culturing the described stem cells under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0068] In one embodiment, a method is provided for treating a patient requiring cell therapy, the method comprising (a) administering the composition described above, and (b) exposing the composition to an exogenous factor for activating an inducible RNA polymerase promoter, thereby controlling the cellular immunogenicity of the composition.

[0069] In one embodiment, a composition is provided comprising isolated mammalian cells, which include modifications comprising recombinant nucleic acid sequences encoding a system for the conditional expression of one or more immunosuppressive factors.

[0070] In one embodiment, a composition is provided comprising isolated mammalian cells containing recombinant nucleic acid sequences encoding a system for the conditional expression of one or more immune signaling factors.

[0071] In some embodiments, the expression of one or more immunosuppressive factors can be controlled by exogenous factors. In some embodiments, the expression of one or more immune signaling factors can be controlled by exogenous factors.

[0072] In some embodiments, the system includes an inducible proteolysis system for reducing the protein levels of one or more immunosuppressive factors. In some embodiments, the inducible proteolysis system is selected from the group consisting of small molecule co-blocking (SMASH) systems, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolysis-inducible chimeric molecules, and antibodies for targeted degradation. In some embodiments, the system includes an RNA regulatory system for controllably reducing the RNA levels of one or more immunosuppressive factors. In some embodiments, the RNA regulatory system is selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems. In some embodiments, the RNA regulatory system is controllable by ligand-inducible transcription factors, SynNotch receptors, or ligand-controlled riboswitches.

[0073] In some embodiments, the system includes a DNA regulatory system for reducing the expression level of one or more immunosuppressive factors, selected from the group consisting of tissue-specific promoter expression systems, inducible promoter expression systems, molecularly controlled riboswitch systems, and inducible nuclease-based genome editing systems.

[0074] In some embodiments, the inducible promoter expression system includes a U6Tet promoter and a Tet repressor element.

[0075] In some embodiments, the system includes an inducible protein stabilization system for increasing the protein levels of one or more immune signaling factors.

[0076] In some embodiments, the inducible protein stabilization system includes a ligand-induced protein stabilization system and a small molecule-induced protein stabilization system.

[0077] In some embodiments, the system includes an RNA regulatory system for increasing the RNA levels of one or more immune signaling factors.

[0078] In some embodiments, the RNA regulatory system includes a CRISPR activation (CRISPRa) system.

[0079] In some embodiments, the system includes a DNA regulatory system for increasing the expression level of one or more immune signaling factors.

[0080] In some embodiments, the DNA regulatory system includes one selected from the group consisting of a CRISPR activation (CRISPRa) system, a tissue-specific promoter, an inducible promoter, and a molecularly controlled riboswitch system.

[0081] In some embodiments, the tissue-specific promoter is selected from the group consisting of cardiac cell-specific promoters, hepatocyte-specific promoters, kidney cell-specific promoters, pancreatic cell-specific promoters, nerve cell-specific promoters, immune cell-specific promoters, mesenchymal cell-specific promoters, and endothelial cell-specific promoters.

[0082] In some embodiments, the inductive promoter includes a TetOn system.

[0083] In some embodiments, the molecularly controlled riboswitch system includes a theophylline-controlled riboswitch or a guanine-controlled riboswitch.

[0084] In some embodiments, the inducible nuclease-based genome editing system includes one selected from the group consisting of CRISPR genome editing, inducible TALEN genome editing, inducible ZFN genome editing, and small molecule-enhanced CRISPR-based genome editing, which include an inducible guide RNA targeting one or more immunosuppressive factors.

[0085] In some embodiments, one or more immunosuppressive factors are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0086] In some embodiments, one or more immune signaling factors are selected from the group consisting of beta-2-microglobulin (B2M), MHC class I-related protein A (MIC-A), MHC class I-related protein B (MIC-B), HLA-A, HLA-B, HLA-C, RFXANK, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D.

[0087] In some embodiments, isolated mammalian cells are isolated human cells further comprising the deletion or reduction of expression of MHC class I human leukocyte antigens and / or the deletion or reduction of expression of MHC class II human leukocyte antigens compared to unmodified human cells.

[0088] In some embodiments, isolated human cells further include deletion or reduced expression of CIITA, B2M, and / or NLRC5.

[0089] In some embodiments, isolated human cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0090] A composition is provided which comprises isolated differentiated cells prepared by culturing one of the described stem cells under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0091] In another embodiment, a method for treating a patient requiring cell therapy is outlined, the method comprising (a) administering the outlined composition, and (b) exposing the composition to an exogenous factor for controlling the expression of one or more immunosuppressive factors, thereby controlling the cellular immunogenicity of the composition.

[0092] In one embodiment, a construct is outlined comprising (1) a safety switch gene, (2) a sequence encoding a ribosome skipping sequence and / or a linker, and (3) a low-immunity gene at the 5' to 3' end. In another embodiment, a construct is outlined comprising (1) a low-immunity gene, (2) a ribosome skipping sequence or linker, and (3) a safety switch gene at the 5' to 3' end.

[0093] In some embodiments, the safety switch gene is HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, P The gene is selected from a group consisting of SMA-transformed genes, CD16-transformed genes, and CD30-transformed genes.

[0094] In some embodiments, the ribosome skipping sequence includes a sequence that encodes an IRES sequence or a sequence that encodes a 2A coding sequence.

[0095] In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0096] In some embodiments, the hypoimmune genes are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0097] In some embodiments, the construct further comprises a transcriptional regulatory element operably linked to a safety switch gene and a polyadenylated sequence at the 3' end of a hypoimmune gene, or a transcriptional regulatory element operably linked to a hypoimmune gene and a polyadenylated sequence at the 3' end of a safety switch gene.

[0098] In some embodiments, the transcriptional control element of the construct is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0099] In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0100] In some embodiments, a method is provided for delivering a construct into isolated cells, comprising transducing isolated cells with a lentiviral construct containing one of the constructs outlined, and selecting engineered cells possessing a safety switch gene and a low-immunity gene.

[0101] Isolated cells or populations containing the constructs described are provided herein. In some embodiments, the constructs are introduced into target loci. In some embodiments, the loci are either safe harbor loci selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5, or immune signaling loci selected from the group consisting of other ligands including B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and NKG2D. In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells.

[0102] Furthermore, differentiated cells or populations thereof are also provided, which are prepared by culturing stem cells under differentiation conditions appropriate for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0103] A method is provided for treating a patient in need of cell therapy, comprising administering the patient a described differentiated cell population or a population thereof. A method is also provided for treating a patient, comprising activating a safety switch in a patient who has been previously administered the described differentiated cell population or a population thereof.

[0104] In one embodiment, a construct for homology-directed repair into a safe harbor locus is provided, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of the safe harbor locus; (2) a safety switch introduction gene; (3) a sequence encoding a ribosome skipping sequence and / or a linker; (4) a low-immunity gene; (5) a polyadenylation sequence; and (6) a second homology arm homologous to a second endogenous sequence of the safe harbor locus. In one embodiment, a construct for homology-directed repair into a safe harbor locus is provided, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of an immune signaling locus; (2) a safety switch transgene; (3) a sequence encoding a ribosome skipping sequence and / or a linker; (4) a low-immunity gene; (5) a polyadenylation sequence; and (6) a second homology arm homologous to a second endogenous sequence of an immune signaling locus. In one embodiment, a construct for homology-directed repair into a safe harbor locus is provided, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of the safe harbor locus; (2) a safety switch introduction gene; (3) a sequence encoding a ribosome skipping sequence or linker; (4) an essential cell factor gene; (5) a polyadenylation sequence; and (6) a second homology arm homologous to a second endogenous sequence of the safe harbor locus. In another embodiment, a construct for homology-directed repair into an immune signaling locus is provided, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of the immune signaling locus; (2) a safety switch introduction gene; (3) a sequence encoding a ribosome skipping sequence or linker; (4) an essential cell factor gene; (5) a polyadenylation sequence; and (6) a second homology arm homologous to a second endogenous sequence of the immune signaling locus. In another embodiment, a construct for homology-directed repair into an essential cell factor locus is provided, comprising at its 5' to 3' end: (1) a first homology arm homologous to the first endogenous sequence of the essential cell factor locus, (2) a sequence encoding a linker, (3) a safety switch introduction gene, and (4) a second homology arm homologous to the second endogenous sequence of the essential cell factor locus.

[0105] In some embodiments, the hypoimmune genes are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0106] In some embodiments, essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

[0107] In some embodiments, the safe harbor locus is selected from the group consisting of the AAVS1 locus, CLBYL locus, CXCR4 locus, Rosa26 locus, and CCR5 locus.

[0108] In some embodiments, the immune signaling gene loci are B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAE11L / ULBP6, RAET1N / ULBP3 Selected from the group consisting of , and other ligands for NKG2D.

[0109] In some embodiments, the immune signaling locus is selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, and HLA-E.

[0110] In some embodiments, the ribosome skipping sequence includes a sequence that encodes an IRES sequence or a sequence that encodes a 2A coding sequence.

[0111] In some embodiments, the 2A code sequence is selected from the group consisting of T2A, P2A, E2A, and F2A.

[0112] In some embodiments, the construct allows a targeted nuclease to cleave a safe harbor locus or immune signaling locus, thereby enabling the construct to be recombined into the locus by homology-directed repair.

[0113] In some embodiments, the construct allows a targeted nuclease to cleave an essential cell factor locus, thereby enabling the construct to be recombined into the locus by homology-directed repair.

[0114] In some embodiments, the construct further includes a transcriptional regulatory element located at the 5' end of the safety switch transgene, selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0115] In some embodiments, the safety switch gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD16 gene, and CD30 gene.

[0116] In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0117] Isolated cells or populations comprising safety switch genes and hypoimmune genes integrated into safe harbor loci or immune signaling loci are outlined herein, wherein one of the above constructs is recombined into an endogenous safe harbor locus of the cell, or one of the above constructs is recombined into an endogenous immune signaling locus of the cell. Isolated cells or populations comprising safety switch genes and essential cell factor genes integrated into safe harbor loci or immune signaling loci are outlined herein, wherein one of the above constructs is recombined into an endogenous safe harbor locus of the cell, or one of the above constructs is recombined into an endogenous immune signaling locus of the cell, and the cell or population thereof is unable to express essential cell factors from the endogenous locus.

[0118] In some embodiments, the isolated cells are isolated and engineered human cells, further comprising the deletion or reduction of expression of MHC class I human leukocyte antigens and / or the deletion or reduction of expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further include deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0119] A differentiated cell population or a population thereof is provided, which is prepared by culturing any of the stem cells outlined herein under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0120] Disclosed are methods for treating patients in need of cell therapy, including administering to the patient a population of differentiated cells or a population of differentiated cells outlined. Also disclosed are methods for treating patients, including activating a safety switch in patients who have been pre-administered with the differentiated cells or a population of differentiated cells outlined.

[0121] A homology-independent donor construct is provided, comprising, at the 5' to 3' ends, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) a safety switch transgene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) a hypoimmune gene, (6) a polyadenylation sequence, and (7) a 3' LTR containing a right-side element (RE).

[0122] A homology-independent donor construct is provided, comprising, at the 5' to 3' ends, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) a safety switch transgene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) an essential cell factor gene, (6) a polyadenylation sequence, and (7) a 3' LTR containing a right-side element (RE).

[0123] A homology-independent donor construct is provided, comprising, at the 5' to 3' ends, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) an essential cell factor gene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) a safety switch transgene, (6) a polyadenylation sequence, and (7) a 3' LTR containing a right-side element (RE).

[0124] In some embodiments, the hypoimmune genes are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0125] In some embodiments, essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

[0126] In some embodiments, the construct is configured to be incorporated into a target gene locus in an isolated cell to interfere with the expression of the target gene.

[0127] In some embodiments, the safety switch gene is the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, The genes are selected from a group consisting of horseradish peroxidase genes, iCaspase9 genes, HER1 transgenes, RQR8 transgenes, CD20 transgenes, CCR4 transgenes, HER2 transgenes, CD19 transgenes, MUC1 transgenes, EGFR transgenes, GD2 transgenes, PSMA transgenes, CD16 transgenes, and CD30 transgenes.

[0128] In some embodiments, the target locus is an immune signaling locus selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D. In some embodiments, the target locus is an immune signaling locus selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, and HLA-E. In some embodiments, the target locus is a safe harbor locus selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5.

[0129] Isolated cells or populations containing one of the constructs described herein are outlined herein, the construct being incorporated into an endogenous target gene to interfere with the expression of the target gene in the isolated cells. Isolated cells or populations described herein are unable to express essential cellular factors from endogenous loci.

[0130] In some embodiments, the construct is integrated into the target gene at a nuclease or transposase target site. In some embodiments, one allele of the target gene is disrupted by nuclease or transposase targeting. In some embodiments, both alleles of the target gene are disrupted by nuclease or transposase targeting. In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0131] In some embodiments, differentiated cells or populations thereof are provided herein, prepared by culturing outlined stem cells under differentiation conditions appropriate for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells. In some embodiments, a method is provided for treating a patient in need of cell therapy, comprising administering the differentiated cells or population to the patient. In some embodiments, a method is provided for treating a patient, comprising activating a safety switch in a patient who has been pre-administered differentiated cells or population.

[0132] Isolated cells or populations thereof are provided herein, comprising essential cell factor genes operably ligated to sequences encoding linkers operably ligated to safety switch-introduced genes.

[0133] In some embodiments, essential cellular factors include RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, and proteasome subunit proteins. The linker is selected from the group consisting of proteins and spliceosome subunit proteins. In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0134] In some embodiments, the safety switch gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD30 gene, and CD16 gene.

[0135] In some embodiments, recombinant peptide epitope fusion proteins are provided herein, comprising (1) a low-immunity factor selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms, and (2) a peptide epitope exposed on a surface different from the low-immunity factor, selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

[0136] In some embodiments, recombinant peptide epitope fusion proteins are provided herein, comprising (1) a sequence encoding a low-immunity factor selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms, and (2) a sequence encoding a surface-exposed peptide epitope of a different type from the low-immunity factor, selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

[0137] In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR epitope is recognized by Thomzo The epitope is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0138] In some embodiments, the low-immunity factor and / or peptide epitope is located at the N-terminus of the fusion protein.

[0139] In some embodiments, the protein further comprises a linker located at the N-terminus or C-terminus of the fusion protein, which links the low-immunity factor and the peptide epitope. In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0140] In some embodiments, the sequence encoding the low immunofactor of the construct is located at 5' of the sequence encoding the peptide epitope, and / or the sequence encoding the peptide epitope is located at 5' of the sequence encoding the low immunofactor.

[0141] In some embodiments, the construct further includes a sequence that links a sequence encoding a low-immunity factor and a sequence encoding a peptide epitope, and / or a sequence encoding a linker located at the N-terminus or C-terminus of the fusion protein. In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0142] In some embodiments, the construct further includes a transcriptional regulatory element selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. In some embodiments, the construct further includes a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair. In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0143] Furthermore, a method is provided for delivering a construct into isolated cells, comprising transducing isolated cells with a lentiviral construct containing the construct described herein and selecting engineered cells expressing a recombinant peptide epitope fusion protein. In some embodiments, a method is provided comprising transducing isolated cells with one of the described constructs and selecting isolated cells expressing a recombinant peptide epitope fusion protein encoded by the construct.

[0144] Also provided are isolated cells or populations thereof, including the constructs described herein. In some embodiments, the isolated cells are isolated human cells further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprising deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some embodiments, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0145] Furthermore, differentiated cells or populations thereof, prepared by culturing the described stem cells under differentiation conditions appropriate for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells, are outlined herein.

[0146] In some cases, a method is provided for treating patients requiring cell therapy, which may include administering the patient the differentiated cells or population described. In some cases, differentiated cells or The invention provides a method for treating a patient, which involves administering an antibody that binds to a peptide epitope to a patient who has been pre-treated with that population. In some embodiments, the antibody is mediated by ADCC or CDC.

[0147] In some embodiments, recombinant CD47 internal peptide epitope fusion proteins are described, comprising (1) a human CD47 fragment containing the IgV domain of CD47, (2) a first linker, (3) a heterologous peptide epitope, (4) a second linker, and (5) a human CD47 transmembrane domain, from the N-terminus to the C-terminus.

[0148] In some embodiments, the human CD47 fragment containing the IgV domain includes amino acid residues 1-127 of the human CD47 protein. In some embodiments, the human CD47 transmembrane domain includes amino acid residues 128-348 of the human CD47 protein. In some embodiments, the first and second linkers are selected from any one of the linkers provided in Table 3. In some embodiments, the peptide epitope is selected from the group consisting of the CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLib, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR epitope is recognized by Thomzo The epitope is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0149] In another embodiment, a construct is disclosed comprising, at its 5' to 3' end, (1) a transcriptional regulatory element, (2) a sequence encoding a human CD47 fragment including the IgV domain of CD47, (3) a first linker, (4) a sequence encoding a peptide epitope, (5) a second linker, and (6) a sequence encoding a human CD47 fragment including a transmembrane domain and a C-terminus.

[0150] In some embodiments, the human CD47 fragment containing the IgV domain contains amino acid residues 1-127 of the human CD47 protein. In some embodiments, the human CD47 fragment containing the transmembrane domain and C-terminus contains amino acid residues 128-348 of the human CD47 protein. In some embodiments, the first and second linkers are selected from any one of the linkers provided in Table 3. In some embodiments, the peptide epitopes encoded by the sequence of (4) of the construct are the CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope. The group consists of topes. In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLib, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR epitope is recognized by Thomzo The epitope is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0151] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0152] In some embodiments, the construct further includes a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair. In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0153] A method is provided for delivering a construct into isolated cells, comprising transducing isolated cells with a lentiviral construct containing the construct and selecting engineered cells expressing a CD47 internal peptide epitope fusion protein. In some embodiments, the method comprises transducing isolated cells with one of the described constructs and selecting isolated cells expressing a CD47 internal peptide epitope fusion protein encoded by the construct. Also provided are isolated cells or populations thereof containing the construct.

[0154] In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0155] Furthermore, differentiated cells or populations thereof, prepared by culturing the described stem cells under differentiation conditions appropriate for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells, are outlined herein.

[0156] In some cases, a method is provided for treating a patient in need of cell therapy, comprising administering the patient a described differentiated cell population or a population thereof. In some cases, a method is provided for treating a patient who has been pre-administered differentiated cell population or a population thereof, comprising administering the patient an antibody that binds to a peptide epitope. In some embodiments, the antibody is mediated by ADCC or CDC.

[0157] In one embodiment, a construct is provided comprising (1) a transcriptional regulatory element, (2) an essential cell factor gene, (3) a post-transcriptional or post-translational regulatory element, and (4) a polyadenylated sequence.

[0158] In some embodiments, essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

[0159] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. In some embodiments, the post-transcriptional regulatory element is an RNA regulatory system selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems. In some embodiments, the post-translational regulatory element is an inducible proteolysis system selected from the group consisting of small molecule co-blocking (SMASH) systems, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolysis-inducing chimeric molecules, and antibodies for targeted degradation.

[0160] Also provided are isolated cells containing recombinant essential cell factors under the control of post-transcriptional or post-translational regulatory elements, in which endogenous essential cell factor genes are inactivated and the expression of recombinant essential cell factors is regulated by exogenous factors. In some embodiments, the essential cell factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins. In some embodiments, the post-transcriptional regulatory element is an RNA regulatory system selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems. In some embodiments, the post-translational regulatory element is an inducible proteolytic system selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolytic chimeric molecules, and antibodies for targeted degradation. In some embodiments, the isolated cells are autologous human cells or allogeneic human cells.

[0161] In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and selected from the group consisting of stem cells and differentiated cells.

[0162] In yet another embodiment, a bisistronic construct is provided herein, comprising at its 5' to 3' end (1) a transcriptional regulatory element, (2) a sequence encoding a surface-exposed peptide epitope, (3) a ribosome skipping sequence, and (4) a sequence encoding a low-immunity factor. In some embodiments, the low-immunity factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms. In some embodiments, the surface-exposed peptide epitope encoded by the sequence of (2) of the construct is selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR epitope is recognized by Thomzo The epitope is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0163] In some embodiments, the ribosome skipping sequence includes a sequence that encodes an IRES sequence or a sequence that encodes a 2A coding sequence.

[0164] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0165] In some embodiments, the construct further includes a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair. In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0166] In some embodiments, a method for delivering the construct into isolated cells is outlined, comprising transducing isolated cells with a lentiviral construct containing the described construct and selecting engineered cells expressing a low-immunity factor and a peptide epitope. In some embodiments, the method comprises transducing isolated cells with the described construct and selecting isolated cells expressing a low-immunity factor and a peptide epitope, both encoded by the construct. Isolated cells or populations containing the described construct are also provided.

[0167] In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5.

[0168] In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells. In some embodiments, differentiated cells or populations are provided, prepared by culturing the described stem cells under differentiation conditions appropriate for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells. In some cases, a method is provided for treating a patient in need of cell therapy, comprising administering the differentiated cells or population to the patient. In some cases, a method is provided for treating a patient, comprising administering an antibody that binds to a peptide epitope to a patient who has been pre-administered differentiated cells or population. In some cases, the antibody is mediated by ADCC or CDC.

[0169] In one embodiment, pluripotent stem cells are provided, comprising (i) reduced or silencing expression of MHC class I molecules and / or MHC class II molecules, (ii) a safety switch gene, and (iii) a hypoimmune gene, wherein the expression of the safety switch gene modulates the expression of the hypoimmune gene.

[0170] In another embodiment, pluripotent stem cells are provided comprising (i) reduced or silencing of B2M and CIITA expression, (ii) overexpression of CD47, (iii) a safety switch gene, and (iv) a hypoimmune gene, wherein the expression of the safety switch gene modulates the expression of the hypoimmune gene.

[0171] In another embodiment, pluripotent stem cells are provided comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) a safety switch, and (iii) a low-immunity factor, wherein the expression of the safety switch modulates the expression of the low-immunity factor.

[0172] In another embodiment, pluripotent stem cells are provided comprising (i) reduced or silencing expression of B2M and CIITA, (ii) overexpression of CD47, (iii) a safety switch, and (iv) a low-immunity factor, wherein the expression of the safety switch modulates the expression of the low-immunity factor.

[0173] In one embodiment, pluripotent stem cells are provided that (i) reduce or silence the expression of MHC class I molecules and / or MHC class II molecules, and (ii) include low-immunity factors linked to surface-exposed peptide epitopes, the peptide epitopes being selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope, and the low-immunity factors being selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms.

[0174] In one embodiment, pluripotent stem cells are provided that include (i) reduced or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) a low-immunity factor linked to a surface-exposed peptide epitope, wherein the peptide epitope is CD20 epi The group consisting of tops, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope is selected, and the low immunofactors are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms.

[0175] In one embodiment, a construct is provided comprising (1) a safety switch gene, (2) a sequence encoding a ribosome skipping sequence and / or a linker, and (3) an essential cell factor gene at its 5' to 3' end. In some embodiments, a construct is provided comprising (1) an essential cell factor gene, (2) a ribosome skipping sequence or linker, and (3) a safety switch gene at its 5' to 3' end.

[0176] In some embodiments, the safety switch gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD16 gene, and CD30 gene.

[0177] In some embodiments, the ribosome skipping sequence includes a sequence that encodes an IRES sequence or a sequence that encodes a 2A coding sequence.

[0178] In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0179] In some embodiments, the hypoimmune genes are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0180] In some embodiments, the construct further comprises a transcriptional regulatory element operably linked to a safety switch gene and a polyadenylated sequence at the 3' end of a hypoimmune gene, or a transcriptional regulatory element operably linked to a hypoimmune gene and a polyadenylated sequence at the 3' end of a safety switch gene.

[0181] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0182] In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0183] A method for delivering a construct into isolated cells is outlined, comprising transducing isolated cells with a lentiviral construct containing the described construct, and selecting engineered cells possessing a safety switch gene and a low-immunity gene. In some embodiments, the method involves transducing isolated cells with the described construct (e.g., a lentiviral construct) and selecting isolated cells possessing the safety switch gene and low-immunity gene of the construct. A method is provided which includes selecting cells. Also provided are isolated cells or populations thereof containing any one of the constructs described.

[0184] In some embodiments, the construct is introduced into a target locus. In some embodiments, the target locus is selected from a group of safe harbor loci selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5, and immune signaling loci selected from the group consisting of other ligands including B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and NKG2D.

[0185] In some embodiments, the isolated cells are engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some embodiments, the stem cells are selected from embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0186] In another embodiment, differentiated cells or populations thereof are disclosed, which are prepared by culturing any stem cell under differentiation conditions suitable for the differentiation of the stem cell into a cell type selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0187] In some embodiments, a method is provided for treating a patient in need of cell therapy, comprising administering the patient a disclosed differentiated cell population or a population thereof. In some embodiments, a method is provided for treating a patient who has been pre-administered the disclosed differentiated cell population or a population thereof, comprising activating a safety switch in the patient.

[0188] In another embodiment, a recombinant peptide epitope fusion protein is provided, comprising (1) an essential cell factor and (2) a peptide epitope exposed on the surface of a different species from the essential cell factor.

[0189] In some embodiments, essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, spliceosome subunit proteins, and their membrane-bound forms.

[0190] In some embodiments, the peptide epitope is selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

[0191] In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars; and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.6 The 0C3-RLIc epitope is recognized by a therapeutic antibody selected from the group consisting of these biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars; or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0192] In some embodiments, the essential cell factor is located at the N-terminus of the fusion protein. In some embodiments, the peptide epitope is located at the N-terminus of the fusion protein. In some embodiments, the protein further includes a linker connecting the essential cell factor and the peptide epitope. In some embodiments, the protein further includes a linker located at the N-terminus of the peptide epitope. In some embodiments, the linker is selected from any one of the linkers provided in Table 3.

[0193] In another embodiment, a construct encoding a recombinant peptide epitope fusion protein is provided, comprising (1) a sequence encoding an essential cell factor, and (2) a sequence encoding a surface-exposed peptide epitope of a different type from the essential cell factor.

[0194] In some embodiments, essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, spliceosome subunit proteins, and their membrane-bound forms. In some embodiments, peptide epitopes are selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR epitope is recognized by Thomzo The epitope is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars; the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0195] In some embodiments, the sequence encoding the essential cell factor is located at 5' of the sequence encoding the peptide epitope.

[0196] In some embodiments, the sequence encoding the peptide epitope is located at 5' of the sequence encoding the essential cell factor.

[0197] In some embodiments, the construct further includes a sequence encoding a linker that connects a sequence encoding an essential cell factor and a sequence encoding a peptide epitope. In some embodiments, the construct further includes a sequence encoding a linker located at the N-terminus or C-terminus of the fusion protein. In some embodiments, the linker is selected from any one of the linkers provided in Table 3. In some embodiments, the construct includes a transcriptional regulatory element selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0198] In some embodiments, the construct further includes a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair. In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0199] In some embodiments, a method for delivering the construct into isolated cells is outlined, comprising transducing isolated cells with a lentiviral construct containing the described construct and selecting engineered cells expressing a recombinant peptide epitope fusion protein. In other embodiments, the method comprises transducing isolated cells with a lentiviral construct containing the described construct and selecting isolated cells expressing a recombinant peptide epitope fusion protein in the construct. In some embodiments, the isolated cells or population contain the constructs referred to herein.

[0200] In some embodiments, the isolated cells are isolated and engineered human cells, further comprising deletion or reduced expression of MHC class I human leukocyte antigens and / or deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further comprise deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0201] In some embodiments, differentiated cells or populations are provided, prepared by culturing described stem cells under differentiation conditions suitable for the differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells. In some cases, a method is provided for treating a patient in need of cell therapy, comprising administering the differentiated cells or populations to the patient. In some cases, a method is provided for treating a patient, comprising administering an antibody that binds to a peptide epitope to a patient who has been pre-administered differentiated cells or populations. In some cases, the antibody is mediated by ADCC or CDC.

[0202] From the 5' to the 3' end, (1) a first homology arm homologous to the first endogenous sequence of the safe harbor locus, (2) a transcriptional regulatory element, (3) an HSVtk safety switch transgene, (4) a sequence encoding a ribosome skipping sequence and / or a linker, and (5) C A construct for homology-directed repair into a safe harbor locus is provided, comprising (6) a polyadenylated sequence and (7) a second homology arm homologous to the second endogenous sequence of the safe harbor locus. A construct for homology-directed repair into a safe harbor locus is provided, comprising at the 5' to 3' end: (1) a first homology arm homologous to the first endogenous sequence of the immune signaling locus, (2) a transcriptional regulatory element, (3) an HSVtk safety switch transgene, (4) a sequence encoding a ribosome skipping sequence and / or a linker, (5) a CD47 hypoimmune gene, (6) a polyadenylated sequence and (7) a second homology arm homologous to the second endogenous sequence of the immune signaling locus.

[0203] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. In some embodiments, the construct further includes a vector backbone for lentiviral expression.

[0204] Isolated cells or populations containing safety switch transgenes and hypoimmune genes integrated into safe harbor loci or immune signaling loci are provided, wherein the constructs described herein are recombined within the endogenous safe harbor loci of the isolated cells or within the endogenous target loci of the isolated cells.

[0205] In some embodiments, the isolated cells further include deletion or reduced expression of MHC class I human leukocyte antigens and / or MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated cells further include deletion or reduced expression of CIITA, B2M, and / or NLRC5. In some embodiments, the isolated cells are low immunogenic and are stem cells. In some cases, the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells.

[0206] Differentiated cells or populations thereof, prepared by culturing the described stem cells under differentiation conditions suitable for differentiation into any of the pancreatic stem cells, are provided herein. In some embodiments, the pancreatic cells are beta islet cells.

[0207] A method for treating patients requiring cell therapy is outlined, which includes administering the disclosed differentiated cells or population to the patient and activating a safety switch in patients who have been previously administered the differentiated cells or population as described.

[0208] Detailed descriptions of low immunogenic cells, methods for producing them, and methods for using them can be found in WO2016183041, filed on 9 May 2015, and WO2018132783, filed on 14 January 2018, and these disclosures, including sequence listings and figures, are incorporated herein by reference in their entirety.

[0209] Other purposes, advantages, and embodiments of this technology will become apparent from the following detailed description. [Brief explanation of the drawing]

[0210] [Figure 1] The data from HEK293 cell lines engineered to express mouse CD47, showing various knockdown efficiencies of different shRNA constructs, are illustrated. Inducible shRNAs regulated by the TetO system were used for the downregulation of exogenous CD47. [Figure 2] A schematic diagram of the Small Molecular-Weight Co-blocking (SMASH) system for inducing CD47 degradation is provided (see Hannah et al., Nature Chemical Biology 11637-638 (2015)). SMASH is a system that effectively blocks the expression of CD47 protein fused to a SMASH tag using elements of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease and NS4A protein, with a clinically tested HCV protease inhibitor. In the absence of the protease inhibitor (asunaprevir), the cryptic degron sequence is excised, resulting in an unmodified gene product. The addition of asunaprevir inhibits the NS3 protease, causing degradation of the newly synthesized CD47 protein fused to the degron sequence. [Figure 3] Figures A and B provide schematic diagrams of donor templates for homology-directed repair (HDR) into the AAVS1 genome safe harbor locus. Figure A shows a cassette containing the EF1a core promoter (EFS) and a SMASH tag fused to the human CD47 gene, inserted between two 1000 bp homology arms into the AAVS1 genome safe harbor locus (hereinafter referred to as the AAVS1-EFS-SMASH-CD47-AAVS1 donor template). Figure B shows that CD47 overexpression is achieved by knocking in the AAVS1-EFS-SMASH-CD47-AAVS1 cassette into the AAVS1 genome safe harbor locus. [Figure 4] This provides a summary of studies to assess the SMASH system's ability to promote CD47 degradation. (Top) Schematic diagram of an expression construct containing the EFS-SMASH-CD47 expression cassette. This expression construct contains an EF1a core promoter (EFS) operably linked to the human CD47 gene fused to a nucleic acid encoding the SMASH tag. (Bottom) iPSCs transduced to either the EFS-SMASH-CD47 expression cassette or a control EFS-CD47 expression cassette were assessed for CD47 expression for 48 hours in the presence of different concentrations of asunaprevir. [Figure 5] A schematic diagram of a ligand-induced degradation (LID) system for inducing CD47 degradation is provided (see Bonger et al., Nat Chem Biol. 7(8):531-537 (2012)). In the LID system, the target protein (POI, e.g., CD47) is fused to the LID domain. The LID domain contains FK506 and rapamycin-binding protein (FKBP), as well as peptide degron fused to the C-terminus of FKBP. FKBP is an enzyme with cis / transprolyl isomerase activity and can be active against a wide range of substrate polypeptides. Peptide degron can bind to the FKBP active site and, when sequestered at the active site, is undetectable by cytodegradation proteins, thus becoming a hidden degron. In the absence of low molecular weight shield-1, the POI-LID fusion protein is stable. However, in the presence of Shield-1, Shield-1 tightly binds to FKBP, thereby replacing peptide degron and inducing rapid degradation of LID and any fusion partner protein (e.g., CD47). [Figure 6] This provides a summary of studies to assess the ability of the LID system to promote CD47 degradation. (Top) Schematic diagram of the expression construct, including the EFS-CD47-LID expression cassette used in the studies. This expression construct contains an EF1a core promoter (EFS) operably linked to the human CD47 gene fused to the nucleic acid encoding the LID domain. (Bottom) Summary of experiments in which iPSCs were transduced with either the EFS-CD47-LID expression cassette or a control EFS-CD47 expression cassette and assessed for CD47 expression for 24 hours in the presence of different concentrations of Shield-1. [Figure 7] This provides a schematic diagram of low-immunity cells, in which an activated safety switch causes cells that no longer express the resulting low-immunity factors to be recognized by immune cells and eliminated by the immune system. [Figure 8]A-D schematically illustrate how hypoimmune cells can be manipulated to have hypoimmunity through modification via cell targeting (A), protein regulation (B), RNA regulation (C), and DNA regulation (D). Protein regulation, RNA regulation, and DNA regulation can be inducible, thereby generating inducible hypoimmune cells. [Figure 9] This outlines how the expression of immunosuppressive factors is regulated by controlled degradation or knockdown. See, for example, Liang, Qin, et al. “Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.” Nature 563.7733(2018):701. [Figure 10] This study demonstrates that cells transduced with a lentiviral vector containing an inducible shRNA targeting exogenous CD47 at an MOI greater than 0.3 showed efficient knockdown of CD47. [Figure 11] The architecture of a codependent safety switch-low immune molecule construct is illustrated. Co-expression of the safety switch and low immune molecule is achieved by the expression of a polycistronic transcript in which the low immune molecule and safety switch are separated by a ribosome skipping sequence such as IRES or a 2A self-cleaving peptide. Cassette expression is controlled by a promoter for genome-independent transcriptional regulation or by a splice acceptor to enable payload control by an endogenous promoter following target integration. [Figure 12] This paper schematically illustrates the targeted genome integration of a CD47-HSVtk fusion construct into the B2M locus for the simultaneous disruption of B2M and expression of a safety switch cassette. HSVtk and CD47 are linked via a P2A self-cleaving peptide within a polycistronic cassette. Homological arms complementary to the B2M locus are located on both sides of the cassette, enabling integration via Cas9-induced HDR. [Figure 13]This section outlines the replacement of essential genes with synthetic essential gene-safety switch fusions. By linking the expression of the safety switch to that of the essential gene, switch expression in living cells is ensured. To directly link the expression of the essential gene to the safety switch without relying on the gene product of the endogenous essential gene locus, the cDNA of the essential gene ("SynEssentialGene") is positioned within a cassette and fused to the safety switch. Because the presence of the cassette is necessary and to ensure it is under proper transcriptional regulation, the cassette contains an internal splice acceptor 2A sequence for utilizing the endogenous promoter and a homology arm to the essential gene locus to facilitate its integration. By providing Cas9-sgRNA targeted at the essential gene locus, Cas9-induced HDR enables simultaneous disruption of the gene product at that locus and integration of the cassette, effectively replacing the native gene. [Figure 14] The architecture of the CD47-ADCC / CDC amino-terminus-dependent safety switch is schematically shown. ADCC and CDC function via immune system effector cells that recognize antibodies bound to the extracellular surface of cells. Expression of the antibody-bound epitope is sufficient to activate ADCC / CDC, allowing it to act as a safety switch. By fusing the epitope to hypoimmune molecules such as CD47, a double safety mechanism against the elimination of manipulated hypoimmune cells is encoded. Peptidelic epitopes, such as the CD20 fragment recognized by rituximab, can be linked to extracellular hypoimmune molecules such as CD47. Specifically, fusing the CD20 epitope to CD47 at its N-terminus makes the epitope sterically available for rituximab binding without disrupting CD47 function. This same design can be applied to other hypoimmune molecules. [Figure 15]A schematic diagram of the architecture of a CD47 variant containing an internal CD20 epitope is shown. The CD20 epitope can be directly inserted into CD47 downstream of the IgV domain, placing the epitope between the IgV domain and the newly formed transmembrane domain. The downstream placement of the CD20 epitope downstream of the IgV domain preserves the quaternary structure of IgV. [Figure 16] This provides a summary of studies to assess the ability of cytosine deaminase (demanise) switches to induce cell death in an immune-dependent manner. (Top) Schematic diagram of the EFS-cytosine deaminase (CD)-CD47 bicistronic cassette used in the study. In this cassette, the nucleic acid encoding CD is located upstream of the nucleic acid encoding CD47. A 2A sequence is inserted between the CD nucleic acid and the CD47 nucleic acid to ensure that these two proteins are separate after translation. An EFS promoter is further included in the cassette for the expression of these two proteins. (Center chart) Chart showing the viability of cells transduced with EFS-CD-CD47 and cells transduced with EFS-CD ("CD"; suicide gene only, control to cells transduced with EFS-CD-CD47) in the presence of various concentrations of 5-fluorocytosine (5-FC). (See chart below) CD47 expression in EFS-CD-CD47-transduced cells and CD cells is compared by flow cytometry. [Figure 17]This is a schematic diagram illustrating the components and architecture of a DNA cassette encoding a safety switch or an essential cell factor molecule, either individually or in combination, as a payload. In some embodiments, the payload refers to a safety switch linked to an essential cell factor. Heterogenes containing the cDNA of a safety switch and an essential cell factor contain several components, namely transcriptional regulatory sequences such as ubiquitous promoters or splice acceptors, open reading frames (ORFs) encoding the safety switch or essential cell factor, polyadenylation sequences, and post-transcriptional or post-translational regulatory elements at the amino or carboxyl terminus of the essential gene or payload. Examples of regulatory elements, in particular, may be riboswitches for regulating translation or chemically destabilizable degron motifs existing as fusion proteins with the payload. [Figure 18] This is a schematic diagram illustrating the architecture of a codependent safety switch-essential cell factor construct. Co-expression of the safety switch and essential cell factor molecules is achieved by the expression of a polycistronic transcript in which the essential cell factor and safety switch are separated by a ribosome skipping sequence such as IRES or a 2A self-cleaving peptide. Cassette expression is controlled by a promoter for genomic location-independent transcriptional regulation, or by a splice acceptor to enable payload control by an endogenous promoter following target integration. [Figure 19] This schematic diagram illustrates the integration of a safety switch into an endogenous essential gene locus to ensure safe switch expression, i.e., the configuration of the targeted gene locus. The safety switch is integrated at the C-terminus of an essential gene, such as a ribosome or proteasome gene, to ensure its expression. Upstream of the switch is a linker sequence encoding iRES or 2A, enabling proper separation of the safety switch and the essential gene protein product. The stop codon is moved downstream of the safety switch. [Figure 20]This is a schematic diagram illustrating targeted integration of a payload (e.g., safety switch and essential gene) for co-expression of a safety switch and essential gene within a safe harbor locus. Donor DNA encoding the safety switch payload described above is integrated into a safe harbor locus such as AAVS1 via the activity of a targeted nuclease such as the S. pyogenes Cas9-sgRNA complex. Integration occurs via homology-directed repair (HDR). In some embodiments, the essential gene is knocked out at its endogenous locus. [Figure 21] This schematic diagram illustrates the simultaneous occurrence of disruption of immune-related gene loci and insertion of essential cell factors or safety switches. Disruption of immune-related gene loci such as B2M occurs via Cas9-induced HDR, which incorporates a cassette encoding a safety switch or an essential cell factor cassette. Cassette incorporation removes the endogenous gene product from the frame, resulting in cassette expression. Cassette expression is conferred by a promoter within the cassette or via an endogenous promoter utilizing the splice acceptor 2A sequence. [Figure 22] This is a schematic diagram illustrating the integration of a safety switch or essential cell factor cassette into the carboxyl terminus of an essential gene. As shown in Figure 3, the cassette encoding a safety switch linked to an essential cell factor is constructed to contain two homology arms that target exogenous DNA toward insertion at the carboxyl terminus of the essential gene. Integration occurs via HDR mediated by Cas9-induced DNA repair. [Figure 23] This schematic diagram illustrates targeted homology-independent integration of safety switches or essential cellular factors. Donor DNA encoding safety switch and essential cellular factor payloads lacking homology arms is packaged as a lentiviral genome or transposon to facilitate integration via homology-independent DNA repair processes. Ligation of exogenous DNA occurs at RNA-inducible nuclease or transposase target sequences, and cassette expression is regulated by an endogenous promoter using a splice acceptor 2A sequence. [Figure 24] This is a schematic diagram illustrating targeted gene disruption at essential loci via nuclease activity. RNA-inducible nucleases, such as Cas9, are targeted at the PSMA3 locus or another locus of interest to facilitate the introduction of frameshift mutations that interfere with the proper transcription or translation of protein products. In some embodiments, the function of this switch (e.g., delivery of a safety switch and co-expression construct to a safe harbor locus, an immune signaling locus, or delivery using lentiviral incorporation) requires the inactivation of all copies of the essential gene at the endogenous locus so that the survival of the manipulated cell depends on the expression of the essential gene from the safe harbor locus. The lower panel illustrates strategies for inactivating endogenous loci by introducing double-strand breaks using CRISPR. Repair of these breaks by NHEJ or MMEJ leads to insertions or deletions that inactivate the essential gene at the endogenous locus. [Figure 25] This schematic diagram illustrates the incorporation of post-transcriptional or post-translational regulatory elements at the amino or carboxyl terminus of essential genes. This construct acts as a safety switch by providing exogenous control over the expression of essential genes. [Modes for carrying out the invention]

[0211] I. Introduction Hypoimmune pluripotent stem cells (also referred to herein as "HIP cells") and their differentiated cells, which are engineered to express immune regulator proteins and avoid rejection by the host immune system, hold great promise for allogeneic cell therapy. Introducing safety switches to regulate the activity of such cells upon administration to recipients is a crucial technique for improving the safety of these cell therapies. Embodiments of safety switches based on the control of immunosuppressive factor (e.g., hypoimmune factor) expression in engineered cells are described herein. Methods for controlling the expression of immunosuppressive factors at the protein, RNA, or DNA level, thereby functioning as safety switches for cells (e.g., conditional or inducible expression systems), are provided herein. Also provided are pluripotent stem cells and their derivatives, including modifications for the conditional expression of immunosuppressive factors responsive to exogenous signals such as small molecules or biological agents.

[0212] A key characteristic of HIP cells is the expression of immunosuppressive factors that function to suppress the host cell's immune response to the transplanted cell population. In one embodiment, this safety switch is based on the controllable expression of CD47. CD47 is a component of the innate immune system that functions as a "don't eat me" signal as part of the innate immune system to block phagocytosis by macrophages. In other embodiments, this safety switch...

[0213] Conditionally or inducibly hypoimmunogenic cells (e.g., conditionally hypoimmunogenic pluripotent cells) representing a practical source for any transplantable cell type are provided herein. Such cells are protected from adaptive and innate rejection upon administration to a recipient by conditionally expressing one or more immunogenic factors. The expression of such immunogenic factors is controlled by the activity of a conditional expression system. Non-limiting examples of conditional expression systems include inducible proteolytic systems, inducible RNA regulatory systems, and inducible DNA regulatory systems.

[0214] In some embodiments, the low immunogenic cells outlined herein are not subject to rejection by innate immune cells before induction of the conditional expression system. In some cases, the low immunogenic cells are not sensitive to NK cell-mediated lysis before induction of the conditional expression system. In some cases, the low immunogenic cells are not sensitive to macrophage phagocytosis before induction of the conditional expression system. In some embodiments, the low immunogenic cells outlined herein are subject to rejection by innate immune cells when the conditional expression system is induced. In some cases, the low immunogenic cells are sensitive to NK cell-mediated lysis when the conditional expression system is induced. In some cases, the low immunogenic cells are not sensitive to macrophage phagocytosis when the conditional expression system is induced. In some embodiments, the low immunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into recipients with little to no need for immunosuppressants. Such low immunogenic cells retain cell-specific properties and characteristics at the time of transplantation.

[0215] In some embodiments, stem cells and / or differentiated derivatives thereof are provided herein that conditionally avoid immune rejection in MHC-incompatible allogeneic recipients. In some cases, differentiated cells produced from the stem cells outlined herein conditionally avoid immune rejection when administered to (e.g., transplanted or grafted) an MHC-incompatible allogeneic recipient. In other words, stem cells and / or differentiated cells derived from such stem cells are hypoimmunogenic in the absence of exogenous factors that control the activity of a conditional expression system targeting exogenous immunogenic factors expressed by such cells. In the presence of exogenous factors, the exogenous immunogenic factors are downregulated or degraded according to the conditional expression system. Thus, stem cells and / or differentiated cells derived from such stem cells are no longer hypoimmunogenic. In some cases, the cells do not have reduced immunogenicity (e.g., at least 2.5% to 99% lower immunogenicity) compared to wild-type or unmanipulated cells. In some cases, these cells are immunogenic. In other words, such cells are immunogenic to the recipient and are therefore eliminated by the recipient's immune system and / or become targets for cell death.

[0216] In some embodiments, the stem cells described herein retain the potential and differentiation capabilities of pluripotent stem cells.

[0217] II. Definition As used herein, the term “safety switch” refers to a system for controlling the expression of a gene or protein of interest that, when downregulated or upregulated, causes cell elimination or death, for example, through recognition by the host’s immune system. Safety switches can be designed to be activated by exogenous molecules in the event of an adverse clinical event. Safety switches can be manipulated by controlling expression at the DNA, RNA, and protein levels. Safety switches include proteins or molecules that enable the regulation of cellular activity in response to an adverse event. In one embodiment, the safety switch is a “lethal switch” expressed in an inactive state, which is lethal to cells expressing the safety switch when activated by an externally supplied selective agent. In one embodiment, the safety switch gene is cis-acting on the gene of interest in the construct. Activation of the safety switch causes the cell to kill itself, or itself and adjacent cells, by apoptosis or necrosis.

[0218] When used herein to characterize cells, the term “low immunogenicity” generally means that such cells are less likely to elicit an immune rejection response from the recipient of such cells. For example, compared to unmodified or unaltered wild-type cells, such low immunogenic cells are less likely to elicit an immune rejection response from the recipient of such cells by approximately 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 97.5%. There is a 99% or greater chance of rejection. In some embodiments, genome editing technology is used to regulate the expression of MHC I and MHC II genes, thereby generating low immunogenic cells. In some embodiments, low immunogenic cells avoid immune rejection in MHC-incompatible allogeneic recipients. In some cases, differentiated cells produced from low immunogenic stem cells outlined herein avoid immune rejection when administered to (e.g., transplanted or grafted) MHC-incompatible allogeneic recipients. In some embodiments, low immunogenic cells are protected from rejection by T cell-mediated adaptive immunity and / or rejection by innate immune cells.

[0219] The low immunogenicity of cells can be determined by evaluating 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 effects of low immunogenic cells on T cell proliferation, T cell activation, T cell killing, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, low immunogenic cells and their derivatives undergo reduced killing by T cells and / or NK cells when administered to a subject. In some cases, the cells and their derivatives show reduced macrophage phagocytosis compared to unmodified or wild-type cells. In some embodiments, low immunogenic cells induce a reduced or attenuated immune response in the recipient subject compared to the corresponding unmodified wild-type cells. In some embodiments, low immunogenic cells are non-immunogenic or do not induce an immune response in the recipient subject.

[0220] As used herein, the term “essential cellular factors” refers to proteins or molecules that are necessary for cell survival and / or cell proliferation. For further explanations of the addition of essential cellular factors or essential genes, see, for example, Kabir et al., PloS One, 2017, 12, 5 e0178273; Hart et al., G3, 2017, 7, 2719-2727; Mair et al., Cell Reports, 2019, 27, 599-615; Wang et al., Science, 2015, 350(6264), 1096-1101; Yilmaz et al., Nat Cell Biol, 2018, 20, 610-619; Liu et al., Aging, 2019, 11(12):4011-4031; Ihry et al., Cell Reports, 2019, 27, 616-630; and Bertomeu et al., Mol Cell This can be found in Biol, 2018, 38(1):e00302-17, and Hart et al., Cell, 2015, 163, 1515-1526.

[0221] As used herein, “immunosuppressive factor” or “immune regulatory factor” includes low-immunity factors and, in some cases, also includes complement inhibitors. As used herein, the terms “immunosuppressive factor” and “low-immunity factor” are interchangeable.

[0222] As used herein, "immune signaling factors" may refer to molecules, proteins, peptides, etc., that activate immune signaling pathways.

[0223] As used herein, “inducible expression system” refers to gene expression that can be controlled or induced by ligands, small molecules, peptides, factors, drugs, etc. In some cases, a conditional gene expression system can turn transcription on or off in the presence of ligands, small molecules, peptides, factors, drugs, etc. In some cases, a conditional gene expression system can activate a proteolytic pathway in response to the presence of ligands, small molecules, peptides, factors, drugs, etc.

[0224] As used herein, “degron element” refers to a protein subunit that controls protein degradation. In some cases, degrons contain a sequence of amino acids that provides a degradation signal leading to the cellular degradation of a polypeptide. Degrons can promote the degradation of a bound polypeptide via either the proteasome pathway or the autophagy-lysosome pathway. In a fusion protein, the degron must be operably ligated to the polypeptide of interest, but it does not need to be contiguous with it, as long as the degron still functions to guide the degradation of the polypeptide of interest. Preferably, the degron induces rapid degradation of the polypeptide of interest. For discussions on degrons and their functions in protein degradation, see, for example, Kanemaki et al. (2013) Pflugers Arch. 465(3): 419-425, Erales et al. (2014) Biochim Biophys Acta 1843(1): 216-221, Schrader et al. (2009) Nat. Chem. Biol. 5(11): 815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9): 679-690, Tasaki et al. (2007) Trends Biochem Sci. 32(11): 520-528, Meinnel et al. (2006) Biol. Chem. 387(7): 839-851, and Kim et al. (2013) Autophagy See 9(7):1100-1103, Varshaysky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278, the contents of which are incorporated herein by reference in their entirety.

[0225] As used herein, a “safe harbor locus” refers to a gene locus that enables the safe expression of a transgene or exogenous gene. Examples of “safe harbor” loci include the CCR5 gene, CXCR4 gene, PPP1Rl2C (also known as AAVS1) gene, albumin gene, and Rosa gene.

[0226] 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 means. "Normal presence within a cell" is determined for specific developmental stages and environmental conditions of the cell. Therefore, for example, a molecule present only during the embryonic development of a neuron is an exogenous molecule for adult neuronal cells. Exogenous molecules may include, for example, a functional version of a dysfunctional endogenous molecule, or a dysfunctional version of a normally functioning endogenous molecule.

[0227] Exogenous molecules or factors may, in particular, be low-molecular-weight molecules produced by combinatorial chemistry processes, or high-molecular-weight molecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes comprising one or more of the above molecules. Nucleic acids include DNA and RNA, which may be single-stranded or double-stranded, linear, branched, or cyclic, and of any length. Nucleic acids include nucleic acids capable of forming double helixes, as well as triple-stranded nucleic acids. See, for example, U.S. Patents 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyralases, and helicases.

[0228] Exogenous molecules or constructs may be molecules of the same type as endogenous molecules, such as exogenous proteins or nucleic acids. In such cases, the exogenous molecules are introduced into the cell at higher concentrations than the endogenous molecules within the cell. In some cases, exogenous nucleic acids are the genome of the infecting virus. This may include plasmids or episomes introduced into cells, or chromosomes that are not normally present in cells. Methods for introducing exogenous molecules into cells 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 bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0229] For the purposes of this disclosure, “gene” includes the DNA region that codes for a gene product, and all DNA regions that control the production of the gene product (whether such regulatory sequences are adjacent to the coding sequence and / or transcribed sequence). Therefore, 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 regulatory regions.

[0230] "Gene expression" refers to the conversion of information contained in a gene into a gene product. A gene product can be the direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by the translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristyrylation, and glycosylation.

[0231] "Regulation" of gene expression refers to a change in the level of gene expression. Regulation of expression may include, but is not limited to, gene activation and gene repression. Regulation may also be complete (i.e., gene expression is either completely inactivated or activated to levels above wild-type) or partial (gene expression is either partially reduced or partially activated to some percentage of wild-type levels).

[0232] The terms “operationally linked” or “operably linked” are used interchangeably with respect to the juxtaposition of two or more components (such as sequence elements) arranged to allow for the possibility that both components function correctly and that at least one component may mediate a function on at least one of the other components. Exemplary, a transcriptional regulatory sequence, such as a promoter, is operationally linked to a coding sequence if it controls the transcription level of the coding sequence in response to the presence or absence of one or more transcription factors. Transcriptional regulatory sequences are generally operationally linked to coding sequences in cis, but do not need to be directly adjacent. For example, enhancers are transcriptional regulatory sequences that are operationally linked to a coding sequence, even if they are not contiguous.

[0233] 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 induce the expression of a gene of interest and transfer a gene sequence into a target cell. Thus, this term includes cloning and expression vehicles, as well as embedded vectors. Methods for introducing vectors or constructs into cells 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 bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0234] As used herein, “pluripotent stem cells” have the potential to differentiate into any of three germ layers: endoderm (e.g., stomach wall, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, genitourinary tract tissues), or ectoderm (e.g., epithelial tissue and nervous system tissue). As used herein, the term “pluripotent stem cells” also encompasses “induced pluripotent stem cells” or “iPSCs,” which are a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parent cells include somatic cells that have been reprogrammed by various means to induce a pluripotent, undifferentiated phenotype. Such “iPS” or “iPSC” cells can be produced by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for inducing iPS cells are known in the art and are further described below (see, 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, “hiPSC” refers to human induced pluripotent stem cells.

[0235] The "HLA" or "Human Leukocyte Antigen" complex is a gene complex that codes for major histocompatibility complex (MHC) proteins in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two types of MHC, "HLA-I" and "HLA-II," class I and class II. HLA-I includes three proteins, HLA-A, HLA-B, and HLA-C, which present peptides from inside the cell, and antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with β-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens to T lymphocytes from outside the cell. This stimulates CD4+ T cells (also known as helper T cells). It should be understood that the use of either "MHC" or "HLA" is not intended to be limiting, as it depends on whether the gene originates from a human (HLA) or mouse (MHC) gene. Therefore, when it relates to mammalian cells, these terms may be used interchangeably herein.

[0236] The terms “to treat,” “to treat,” and “to treat” 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 cells or cell populations in which a target polynucleotide sequence (e.g., B2M) has been ex vivo modified according to the methods described herein. The individual is typically diseased or injured, or at increased risk of disease compared to the mean member of the population, and requires such treatment, care, or management.

[0237] As used herein, the terms “to treat” and “treatment” mean administering to a subject an effective amount of cells in which a target polynucleotide sequence has been ex vivo modified according to the method herein, such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this technology, beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, whether detectable or undetectable; attenuation of the severity of the disease; a stabilized (i.e., non-worsening) disease state; delay or slowing of disease progression; improvement or temporary relief of the disease state; and remission (whether partial or complete). To treat may mean extending survival compared to the survival expected without treatment. Therefore, Those skilled in the art will recognize that while treatment may improve the condition, it may not be a complete cure for the disease. As used herein, the term “treatment” includes prevention. Alternatively, treatment is “effective” if it reduces or halts the progression of the disease. “Treatment” may also mean extending survival compared to the survival expected without treatment. Those who require treatment include those already diagnosed with a disorder related to the expression of polynucleotide sequences, as well as those who may develop such a disorder due to genetic susceptibility or other factors.

[0238] "Treatment" or "prevention" of a disease or disorder means delaying or preventing the onset of such disease or disorder, or reversing, mitigating, improving, inhibiting, slowing, or stopping the progression, aggravation, or deterioration of the condition associated with such disease or disorder, or the progression of its severity. In one embodiment, the symptoms of the disease or disorder are mitigated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0239] As used herein, the terms “administer,” “introduce,” and “transplant” are used interchangeably in the context of the placement of cells, e.g., cells described herein, including a target polynucleotide sequence modified according to the methods of this technique, into a subject by a method or route that results in at least partial localization of the introduced cells to a desired site. The cells may be directly implanted into a desired site within a subject, or alternatively, administered by any suitable route that results in delivery to the desired site, where at least a portion of the implanted cells or components of the cells remain viable. The survival time of cells after administration to a subject can range from a short period of a few hours, e.g., 24 hours, to a long period of several days or even several years. In some cases, cells may also be administered to a site other than the desired site, such as in the liver or subcutaneously, in a capsule, to maintain the implanted cells at the implantation site and to avoid migration of the implanted cells.

[0240] In additional or alternative embodiments, the Art intends to modify a target polynucleotide sequence using, for example, a TALEN system, in any manner available to those skilled in the art. While examples of methods utilizing CRISPR / Cas (e.g., Cas9 and Cas12a) and TALENs are detailed herein, it should be understood that the Art is not limited to the use of these methods / systems. Other methods known to those skilled in the art, such as targeting B2M to reduce or eliminate expression in target cells, can be utilized herein.

[0241] The methods outlined herein can be used to modify target polynucleotide sequences within cells. This technology is intended to modify target polynucleotide sequences within cells for any purpose. In some embodiments, the target polynucleotide sequence within a cell is modified to produce mutant cells. As used herein, “mutant cell” refers to a cell whose resulting genotype differs from its original genotype. In some cases, a “mutant cell” exhibits a mutant phenotype, for example, when a normally functioning gene is modified using the CRISPR / Cas system of this technology. In other cases, a “mutant cell” exhibits a wild-type phenotype, for example, when the mutant genotype is corrected using the CRISPR / Cas system of this technology. In some embodiments, the target polynucleotide sequence within a cell is modified to correct or repair a gene mutation (for example, to restore a normal phenotype to the cell). In some embodiments, the target polynucleotide sequence within a cell is modified to induce a gene mutation (for example, to disrupt the function of a gene or genomic element).

[0242] In some embodiments, modifications are indels. When used herein, "indel" refers to an indel. A “modification” refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be 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 3. In some embodiments, the modification is a point mutation. As used herein, a “point mutation” refers to a substitution that replaces one of the nucleotides. CRISPR / Cas systems can be used to induce indels or point mutations of any length in a target polynucleotide sequence.

[0243] As used herein, “knockout” includes the deletion of all or part of a target polynucleotide sequence in a manner that impairs the function of the target polynucleotide sequence. For example, knockout can be achieved by modifying the target polynucleotide sequence by inducing an indel in the target polynucleotide sequence at a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Those skilled in the art will readily understand, based on the details described herein, how to use the CRISPR / Cas system to knock out a target polynucleotide sequence or part of it.

[0244] In some embodiments, the modification results in the knockout of a target polynucleotide sequence or a portion thereof. Knockout of a target polynucleotide sequence or a portion thereof using the CRISPR / Cas system described herein may be useful for a variety of applications. For example, knockout of a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knockout of a target polynucleotide sequence in a cell may be useful to treat or prevent impairments associated with the expression of the target polynucleotide sequence (for example, by ex vivo knockout of a mutant allele in a cell and then introducing those cells containing the knocked-out mutant allele into a target).

[0245] As used herein, “knock-in” means the process of adding gene function to a host cell. In some embodiments, this results in an increase or decrease in the level of the knocked-in gene product, such as RNA or encoded protein. As will be understood by those skilled in the art, this can be accomplished in several ways, including adding one or more additional copies of a gene to a host cell, or increasing the expression of a protein produced by modifying the regulatory components of an endogenous gene. This may be accomplished by modifying a promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0246] In some embodiments, the modification results in a reduction in the expression of the target polynucleotide sequence. The terms “reduce,” “reduced,” “reduced,” and “decrease” are all used herein to generally mean a reduction of a statistically significant amount. However, to avoid doubt, “reduce,” “reduced,” “reduced,” and “decrease” mean a reduction of at least 10% compared to a reference level, e.g., a reduction of 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% compared to a reference level, or a reduction of up to 100% and including this (i.e., a level of absence compared to a reference sample), or any reduction of 10–100%.

[0247] The terms “increased,” “enhance,” “strengthen,” or “activate” are all used herein to generally mean an increase of a statistically significant amount. To avoid ambiguity, the terms “increased,” “enhance,” or “strengthen,” or “activate” refer to an increase of at least 10% compared to a reference level, e.g., at least about 20%, at least about 30%, or at least about 40% compared to a reference level. Or it means an increase of at least approximately 50%, or at least approximately 60%, or at least approximately 70%, or at least approximately 80%, or at least approximately 90%, or an increase of up to 100% or including this, or any increase between 10% and 100%, or an increase of at least approximately 2 times, or at least approximately 3 times, or at least approximately 4 times, or at least approximately 5 times, or at least approximately 10 times, or any increase between 2 times and 10 times or more.

[0248] As used herein, the term “exogenous” is intended to mean that the indicated molecule or indicated polypeptide is introduced into the cell of interest. Polypeptides can be introduced, for example, by incorporating coding nucleic acids into chromosomes or by introducing them into the cell’s genetic material as non-chromosomal genetic material such as plasmids or expression vectors. Thus, when used in relation to the expression of coding nucleic acids, the term refers to the introduction of coding nucleic acids into cells in an expressible form.

[0249] The term "endogenous" refers to a referent molecule or polypeptide that is present within a cell. Similarly, when used in relation to the expression of coding nucleic acids, this term refers to the expression of coding nucleic acids that are contained within the cell and not introduced exogenously.

[0250] In the context of two or more nucleic acid or polypeptide sequences, the term “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned to the maximum extent possible, have a specified percentage of nucleotide or amino acid residues that are identical when measured 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 “identity” percentage may exist across a region of the sequences being compared, for example, across functional domains, or alternatively, across the entire length of the two sequences being compared. For sequence comparison, typically one sequence serves as a reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, the coordinates of any subsequences are specified if necessary, and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence(s) relative to the reference sequence based on the specified program parameters.

[0251] The optimal alignment of sequences for comparison can be determined, 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 similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444 (1988), or by computer implementations of these algorithms (Wisconsin Genetics Software Package, Genetics). This can be done by GAP, BESTFIT, FASTA, and TFASTA (as described by Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al. (see below) for general information).

[0252] One example of a suitable algorithm for determining sequence identity and sequence similarity percentages is the BLAST algorithm, described in Altschul et al, J.Mol.Biol.215:403-410 (1990). Software for performing BLAST analysis is available from the National Center for Biotechnology. It is publicly available via Information.

[0253] The terms “subject” and “individual” are used interchangeably herein and refer to an animal, such as a human, from which cells can be obtained and / or from which treatment with cells (including prophylactic treatment) as described herein is provided. In the case of treatment of an infectious disease, condition, or disease specific to a particular animal, such as a human subject, the term “subject” refers to that particular animal. The terms “non-human animal” and “non-human mammal” are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term “subject” also encompasses any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, a subject is a mammal such as a human, or a domestic mammal such as a dog, cat, or horse, or another mammal such as a production mammal such as a cattle, sheep, or pig.

[0254] It should be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent for the use of exclusive technical terms such as “simply,” “only,” etc., or for the use of “negative” limitations, in relation to the enumeration of elements of the claims. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that are readily decoupled from or combined with any of the features of several other embodiments without departing from the scope or spirit of the Art. Any enumerated method may be carried out in the order of the enumerated events, or in any other logically possible order. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the Art, but representative exemplary methods and materials are described below.

[0255] In this technology, the following terms will be used, and they will be defined as shown below.

[0256] Before further describing the technology, it is to be understood that the technology is not limited to the specific embodiments described herein and thus, as a matter of course, can be various. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the technology is limited only by the appended claims.

[0257] 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 technology belongs. When a range of values is provided, each intervening value, between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated value or intervening value in that stated range, is included within the technology. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, or also within the technology, subject to any specifically excluded limit values within the stated range. When the stated range includes one or both of the limit values, ranges excluding one or both of those included limit values are also included in the technology. A particular range is presented herein with the term "about" preceding a numerical value. The term "about" is used herein to provide literal support for the number itself, as well as for numbers that are near to or approximate the number to which it precedes. When determining whether a number is near to or approximates a specifically recited number, the near or approximate unrecited number may be a number that provides a substantial equivalent of the specifically recited number in the context presented.

[0258] All publications, patents, and patent applications cited herein are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference To the same extent as the case, it is incorporated herein by reference. Further, each publication, patent, or patent application cited is incorporated herein by reference for the purpose of disclosing and describing the subject matter of the invention to which such publication is relevant. The citation of any publication is for the purpose of its disclosure prior to the filing date, and should not be construed as an admission that the technology described herein has the right to precede such a publication based on the prior art. Further, the date of the provided publication may be different from the actual publication date and may need to be independently verified.

[0259] III. MODE FOR CARRYING OUT THE INVENTION A. Method for Conditional HIP Cells and Conditional Downregulation of Immunosuppressive Factors The introduction of a safety switch improves the safety of cell therapies developed using hypoimmunogenic cells (HIP cells). The characteristics of the HIP cells described herein are the inducible expression of one or more immunoregulatory (immunosuppressive) factors. In some embodiments, the immunosuppressive factors (also referred to herein as "hypoimmune factors") include, but are not limited to, CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In certain embodiments, the immunosuppressive factor is CD47. The controllable or inducible expression of the immunosuppressive factor functions to control the recipient's immune response to the transplanted hypoimmunogenic cells.

[0260] Methods for the expression of immunosuppressive factors are described herein that require a mechanism to "turn off" the expression of the immunoregulatory protein in a controlled manner. Also described are HIP cells having controllable expression of one or more immunosuppressive factors. In some cases, the cells overexpress one or more immunosuppressive factors and can be induced to downregulate the expression of one or more immunosuppressive factors. Thus, the cells are no longer hypoimmunogenic and are recognized by the recipient's immune cells towards cell death.

[0261] In some embodiments, the reduced immunity of cells introduced into a recipient is achieved through the overexpression of immunosuppressive molecules, including low-immunity factors and complement inhibitors, and the resulting suppression or disruption of HLA-I and HLA-II loci. These modifications cloak the cells from effector cells of the recipient immune system, such as T cells, B cells, NK cells, and macrophages, which are responsible for eliminating infected, malignant, or non-self cells. Cloaking cells from the immune system allows for the presence and persistence of allogeneic cells in the body. The controlled removal of manipulated cells from the body is essential for patient safety and can be achieved by uncloaking the cells from the immune system. Uncloaking acts as a safety switch and can be achieved through downregulation of immunosuppressive molecules or upregulation of immune signaling molecules. The expression level of any of the described immunosuppressive molecules can be controlled at the protein, mRNA, or DNA level in cells. Similarly, the expression level of any of the described immune signaling molecules can be regulated in cells at the protein, mRNA, or DNA level.

[0262] In some embodiments, one of the described safety switch methods (e.g., protein-level, RNA-level, and DNA-level safety switches) is used to reduce the level of immunosuppressive factors in cells so that lower levels of immunosuppressive factors fall below the threshold level. In some embodiments, the level of immunosuppressive factors in cells is reduced to approximately 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1 time, or 0.5 times below the threshold level of expression. In some embodiments, the level of immunosuppressive factors in cells is reduced to approximately 10 to 5 times, 10 to 3 times, 9 to 1 time, 8 to 1 time, 7 to 0.5 times, 6 to 1 time, 5 to 0.5 times, 4 to 0.5 times, or 3 times below the threshold level of expression. The levels can be reduced to approximately 0.5 times, 2 times to 0.5 times, or 1 time to 0.5 times lower. In some embodiments, the threshold level for immunosuppressive factor expression is established based on the expression of such factors in induced pluripotent stem cells. In some embodiments, the threshold level for immunosuppressive factor expression is established based on the expression level of immunosuppressive factors in corresponding low-immunity cells such as MHC I and MHC II knockout cells or MHC I / MHC II / TCR knockout cells.

[0263] 1. Protein-level regulation In some embodiments, the regulated degradation of immunosuppressive proteins is established by incorporating degron into the amino acid sequence of the immunosuppressive factor, thereby enabling recruitment of the endogenous protein into the metabolic turnover mechanism. Mechanisms for targeted protein degradation include, but are not limited to, recruitment to E3 ligases for ubiquitination and subsequent proteasomal degradation, direct recruitment to the proteasome, and recruitment to lysosomes.

[0264] The fusion of an inducible degron motif with an immunosuppressive molecule allows for exogenous control of molecular stability by stabilizing or destabilizing degron, and consequently the immunosuppressive molecule, through the addition or removal of small molecules.

[0265] In some embodiments, methods for degron-induced proteolysis include, but are not limited to, ligand-induced degradation (LID) using SMASH tags, ligand-induced degradation using Shield-1, ligand-induced degradation using auxin, ligand-induced degradation using rapamycin, peptidolytic degron (e.g., IKZF3-based degron), and proteolysis-inducible chimeric molecules (PROTAC). In some embodiments of ligand-induced degradation methods, the degron tag is held in an inactive conformation but is induced to adopt a conformation that is recognizable by the proteasome when certain molecules, such as Shield-1 molecules, bind to it. For example, Roth et al., Cellular See Molecular Life Sciences, 2019, 76(14), 2761-2777, which is incorporated herein by reference in its entirety. A detailed description of SMASH deglon technology can be found in Hannah and Zhou, Nat Chem Biol, 2015, 11:637-638 and Chung et al., Nat Chem Biol, 2015, 11:713-720, which are incorporated herein by reference in their entirety. A detailed description of LID deglon technology can be found in Bonger et al., Nat Chem Biol, 2011, 7(8):531-7, which are incorporated herein by reference in their entirety.

[0266] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing a construct containing a constitutive promoter operably linked to an inducible degron element operably linked to a gene encoding an immunosuppressive factor. In some embodiments, the construct includes a constitutive promoter operably linked to an inducible degron element, which is operably linked to a nucleic acid sequence encoding a flexible linker, which is operably linked to the gene encoding the immunosuppressive factor. In some embodiments, the construct includes a constitutive promoter operably linked to an immunosuppressive factor, which is operably linked to an inducible degron element, which is linked to a sequence encoding a flexible linker, which is operably linked to an inducible degron element. Thus, degron targets the immunosuppressive factor toward degradation when the cells come into contact with a degron ligand or molecule.

[0267] In some embodiments, the inductive degron element is a low-molecular-weight auxiliary blockade (SMASH) element. The group consists of ligand-inducible degron elements such as gronn elements, Shield-1 responsive degron elements, auxin-responsive degron elements, and rapamycin-responsive degron elements; peptidolytic degron elements; and peptidolytic proteolytic chimeric molecule (PROTAC) elements. In useful embodiments, the ligand-inducible degron element is a small molecule co-blocking (SMASH) degron element, and the exogenous factor for controlling immunogenicity is asunaprevir. In some embodiments, the immunosuppressive gene is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In many embodiments, the immunosuppressive gene is CD47. In some cases, the constitutive promoter of the construct is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. In some cases, the optional flexible linker is (GSG) n (Sequence ID 3), (GGGS) n (Sequence ID 1), and (GGGSGGGS) nA selection is made from the group consisting of (Sequence ID 2), where n is 1 to 10. In some embodiments, the construct is introduced into cells so as to be incorporated into safe harbor loci such as, but not limited to, the AAVS1 locus, CLBYL locus, CXCR4 locus, Rosa26 locus, and CCR5 locus. In some embodiments, the construct is introduced into the AAVS locus in cells via homology-directed recombination. Thus, the construct includes 5' and 3' homology arms specific to the target safe harbor locus. In some embodiments, the construct includes, from the 5' end to the 3' end, a 5' homology arm to the AAVS1 locus, an exogenous constitutive promoter, an inducible degron element, a gene encoding an immunosuppressor, and a 3' homology arm to the AAVS1 locus. In other embodiments, the construct includes, from the 5' end to the 3' end, a 5' homology arm to the AAVS1 locus, an exogenous constitutive promoter, an inducible degron element, a sequence encoding a flexible linker, a gene encoding an immunosuppressor, and a 3' homology arm to the AAVS1 locus. In useful embodiments, the engineered cell includes an exogenous nucleic acid sequence comprising a constitutive promoter operably linked to an inducible degron element operably linked to an optional sequence encoding a flexible linker, which is operably linked to a gene encoding an immunosuppressor. The engineered cell expresses an inducible degron element fused to or linked to an immunosuppressor. In some embodiments, the cell is exposed to a factor or drug, such as a ligand, molecule, peptide, or small molecule, which activates the degron element to degrade the immunosuppressor.

[0268] In some embodiments of peptidolytic degrons, peptide tags are used that confer low-molecular-weight mediated recruitment to E3 ligases. In some embodiments, the peptide tag includes the lymphoid restriction transcription factor IKZF3, which is recruited to the E3 ligase receptor (CRBN) in an immunomodulatory drug (IMiD)-dependent manner, as described in Koduri et al., Proc Natl Acad Sci, 2019, 116(7), 2539-2544 (the entire reference of which is incorporated herein by reference). In certain embodiments, the degron can target immunosuppressive factors toward degradation (e.g., via the ubiquitination pathway) to induce or degrade proteins.

[0269] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing a construct comprising a gene encoding a constitutive promoter, an inducible peptidolytic degron element, and an immunosuppressive factor. In some embodiments, the construct comprises a nucleic acid sequence encoding a constitutive promoter, an inducible peptidolytic degron element, a flexible linker, and an immunosuppressive factor. The method includes genes. Any of the constitutive promoters, immunosuppressants, flexible linkers, and cells described herein are applicable to the method.

[0270] In some embodiments of PROTAC, a bifunctional molecule is used to recruit an immunosuppressive factor into the cellular proteolytic mechanism. In some embodiments, the bifunctional molecule binds to a native or wild-type sequence of an immunosuppressive protein or a modified version of an immunosuppressive protein that expresses a domain that binds to the bifunctional molecule with high affinity. In some embodiments, the bifunctional molecule includes a small molecule or a biological agent (e.g., an antibody or a fragment thereof). See, for example, Burslem et al., Cell Chemical Biology, 2018, 25, 67-77 and Roth et al., Cellular Molecular Life Sciences, 2019, 76(14), 2761-2777, which are incorporated herein by reference in their entirety.

[0271] In some embodiments of bifunctional antibodies, the antibody targets an immunosuppressant and a second endogenous receptor, thereby inducing internal translocation and degradation. Controllable expression of one or more immunosuppressants can be provided using bifunctional antibodies (e.g., chemically reprogrammed bifunctional antibodies), degron-induced proteolysis, inducible RNA regulation, inducible DNA regulation, and inducible expression methods. See, for example, Natsume and Kanemaki, Annu Rev Genet, 2017, 51, 82-102, Burslem See and Crews, Chem Rev, 2017, 117, 11269-11301 and Banik et al., ChemRxiv, 2019, which are incorporated herein by reference in their entirety. In some embodiments, cells expressing an immunosuppressive factor are brought into contact with an antibody that binds to the cell for degradation.

[0272] In some cases, under-immune cells are made available to and eliminated by the immune system by adding antibodies that bind to epitopes on the extracellular surface of the cell. These epitopes may be native to overexpressed immunosuppressive factors, or they may be other epitopes located within the immunosuppressive factor or distinctly located on the extracellular surface. Antibody binding to the surface removes the cell's covering, leading to antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0273] In some embodiments, the ADCC / CDC safety switch epitope is selected from the group consisting of EGFR, CD20, CD19, CCR4, HER2, MUC1, GD2, PSMA, CD30, CD16, and their fragments, derivatives, and variants. In some cases, any of the cells described herein express an epitope selected from the EGFR epitope, CD20 epitope, CD19 epitope, CCR4 epitope, HER2 epitope, MUC1 epitope, GD2 epitope, PSMA epitope, CD30 epitope, or CD16 epitope. In some embodiments, the cell binds to an antibody specific to EGFR, CD20, CD19, CCR4, HER2, MUC1, GD2, PSMA, CD30, or CD16, which induces ADCC / CDC.

[0274] Methods targeting protein-level safety switches as described herein provide means for controllingly reducing the levels of immunosuppressants (e.g., CD47) in engineered cells (e.g., low-immunity cells) as described herein. By using any of the safety-switch methods described herein to reduce the level of immunosuppressants such as CD47 in cells to below a threshold level, the recipient's immune system can initiate an immune response against such cells. In some embodiments, the level of CD47 in engineered cells is reduced by the safety switch to approximately 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, or 0.5-fold below the threshold level of expression. In some embodiments, in engineered cells... The level of CD47 can be reduced to approximately 10 to 5 times, 10 to 3 times, 9 to 1 time, 8 to 1 time, 7 to 0.5 times, 6 to 1 time, 5 to 0.5 times, 4 to 0.5 times, 3 to 0.5 times, 2 to 0.5 times, or 1 to 0.5 times lower than the threshold level of expression. In some cases, the threshold level of CD47 expression is established based on the exogenous expression of CD47 in induced pluripotent stem cells. In other cases, the threshold level of CD47 expression is established based on the expression level of CD47 in corresponding hypoimmune cells such as MHC I and MHC II knockout cells or MHC I / MHC II / TCR knockout cells. In some cases, the level of CD47 is reduced using a degron-based safety switch, such as SMASH degron or LID degron, but is not limited to these. In some embodiments, cells expressing SMASH degron linked to an exogenous CD47 transgene are exposed to low molecular weight asunaprevir (a degron inducer), thereby inducing a reduction in the expression of exogenous CD47 in these cells.

[0275] 2. RNA-level regulation Immunosuppressive factors can be targeted by siRNA or miRNA, thereby causing degradation of the transcripts encoding the factors. siRNA can be provided exogenously or encoded by a gene to provide control over the transcription of inhibitory RNA. siRNA or miRNA can anneal to the transcripts of immunosuppressive factors, resulting in degradation by the RISC complex.

[0276] In some embodiments, methods for downregulating the expression of immunosuppressive factors by inducible RNA control include, but are not limited to, shRNA, inducible siRNA, inducible miRNA, inducible CRISPR interference (CRISPRi), and inducible RNA-targeting nucleases induced by small molecules or biological agents.

[0277] In some embodiments, the method includes shRNA or siRNA that targets the RNA of the immunosuppressive factor. In some cases, the expression of shRNA or siRNA is induced by a small molecule or a biological agent.

[0278] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing a construct comprising an inducible RNA polymerase promoter operably linked to an immunosuppressive factor-targeting shRNA sequence, which in turn is operably linked to a constitutive promoter linked to a transactivator element capable of controlling the inducible RNA polymerase promoter. In some embodiments, the construct comprises a U6Tet promoter, an immunosuppressive factor-targeting shRNA, a constitutive promoter, and a Tet repressor element responsive to tetracycline or a derivative thereof (e.g., doxycycline). In other embodiments, the shRNA eliminates the expression of the immunosuppressive factor. In other embodiments, the shRNA reduces the expression of the immunosuppressive factor by approximately 99% or less, e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 85% or less. In some embodiments, the inducible promoter is a tetracycline-responsive promoter. Any of the constitutive promoters, immunosuppressive factors, and cells described herein can also be applied to the method.

[0279] In many embodiments, the manipulated cells express inducible shRNAs that target immunosuppressive factors. In some embodiments, the cells also express exogenous immunosuppressive factors that mediate the cells' low immunogenicity. In some embodiments, the cells are exposed to factors such as ligands, molecules, peptides, or small molecules, but not limited to them, that activate shRNA expression to degrade the immunosuppressive factors.

[0280] In some embodiments, the method includes a CRISPR interference system (CRISPRi) for targeting the promoter of an immunosuppressant to downregulate its transcription. In some cases, the expression of CRISPRi and / or gRNA targeting the immunosuppressant is induced by a small molecule or biological agent. A detailed description of the CRISPRi method can be found, for example, in Engreitz et al., Cold Spring Harb Perspect Biol, 2019, 11:a035386, which is incorporated herein by reference in its entirety. In some embodiments, the CRISPRi system utilizes a dCas9-repressor fusion protein controlled by a constitutive promoter and an immunosuppressant-specific gRNA under the control of an inducible promoter.

[0281] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing into the cells a first construct containing (i) a constitutive promoter operably linked to a gene encoding an immunosuppressant, (ii) a second construct containing a constitutive promoter operably linked to a gene encoding a Cas9 nuclease or a variant thereof such as a dCas9-repressor fusion protein, and (iii) an inducible RNA polymerase promoter operably linked to a gRNA sequence targeting a sequence encoding an immunosuppressant, such that the gRNA sequence is operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter. In some cases, the first, second, and third constructs are found in a single vector. In some cases, the first, second, and third constructs are found in two vectors.

[0282] In some embodiments, CRISPR-based methods include nucleases for targeting mRNA sequences corresponding to immunosuppressive factors, such as Cas13, Cas7, or Csx1, but are not limited to these. In some cases, the expression of immunosuppressive factor-targeting nucleases and / or gRNAs is induced by small molecules or biological agents.

[0283] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing into the cells a first construct comprising (i) a constitutive promoter operably linked to a gene encoding an immunosuppressive factor, a second construct comprising a constitutive promoter operably linked to a gene encoding a Cas13a nuclease, a variant thereof, or a fusion protein thereof, and (iii) an inducible RNA polymerase promoter operably linked to a gRNA sequence targeting a sequence encoding an immunosuppressive factor, such that the gRNA sequence is operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter.

[0284] In some embodiments, inducible expression systems useful for regulating immunosuppressive factors at the RNA level include, but are not limited to, ligand-inducible transcription system systems, receptor-mediated expression regulatory systems, and ligand-controlled riboswitches. In some embodiments, inducible expression systems include tetracycline-regulated operator systems, synthetic Notch-based (SynNotch) systems (see, e.g., Morsut et al., Cell, 2016, 164:780-791 and Yang et al., Commun Biol, 2020, 3:116), and riboswitches that regulate the expression of immunosuppressive factor genes by ligand-mediated alternative splicing of the resulting pre-mRNA (e.g., aptamers, peptides, or small molecules). Useful riboswitches include sensor and effector regions that sense the presence of a ligand and alter the splicing of the target immunosuppressive factor gene. Detailed descriptions and examples of riboswitch gRNAs can be found, for example, US9,228,207, US9,993,491, and US10,4 21,989, and Seeliger et al., PLoS One, 2012, 7(1):e29266, the contents of which are incorporated herein by reference in their entirety.

[0285] In some embodiments, the levels of immunosuppressive factors such as CD47 in engineered cells are reduced by an RNA-level safety switch to be approximately 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, or 0.5-fold below the threshold level of expression. In some embodiments, the levels of CD47 in engineered cells are reduced to be approximately 10-5-fold, 10-3-fold, 9-1-fold, 8-1-fold, 7-0.5-fold, 6-1-fold, 5-0.5-fold, 4-0.5-fold, 3-0.5-fold, 2-0.5-fold, or 1-0.5-fold below the threshold level of expression. In some cases, the threshold level of CD47 expression is established based on the exogenous expression of CD47 in induced pluripotent stem cells. In other cases, the threshold level for CD47 expression is established based on the expression levels of CD47 in corresponding low-immunity cells such as MHC I and MHC II knockout cells or MHC I / MHC II / TCR knockout cells.

[0286] 3. DNA-level control Transcriptional regulation of immunosuppressive factors using inducible promoters provides the ability to arbitrarily switch their expression on or off by adding or removing small molecules such as doxycycline, though this is not limited to these methods. Gene disruption via targeted nuclease activity can also eliminate the expression of immunosuppressive factors and remove the cell covering.

[0287] In some embodiments, methods for inducible DNA control include, but are not limited to, targeting the DNA sequence of one or more immunosuppressive factors using knockout with tissue-specific promoters, inducible promoters, controllable riboswitches, and inducible nucleases (e.g., inducible CRISPR, inducible TALEN, inducible zinc finger nuclease, inducible homing endonuclease, inducible meganuclease, etc.). In some embodiments, the inducible nuclease includes nucleases whose expression is controlled by the presence of a small molecule. In some embodiments, the inducible nuclease includes nucleases whose delivery of nuclease RNA or protein to cells is controlled by the presence of a small molecule. In some embodiments, nuclease expression is induced by a small molecule or biopharmaceutical. In some embodiments, Cas nuclease and / or guide RNA (gRNA) expression is induced by a small molecule or biopharmaceutical.

[0288] In some embodiments, methods for inducible expression include, but are not limited to, ligand-inducible transcription factor systems (e.g., tetracycline-regulated operator systems), receptor-mediated regulation of expression systems (e.g., SynNotch systems), and ligand-regulated riboswitch systems for regulating mRNA or gRNA activity. A detailed description of inducible expression methods can be found, for example, in Kallunki et al., Cells, 2019, 796 (doi:10.3390 / cells8080796), which is incorporated herein by reference in its entirety.

[0289] In some embodiments, the immunosuppressive factor is expressed in cells using an inducible expression vector. The expression vector may be a viral vector, such as a lentiviral vector, but is not limited to this. In some embodiments, the inducible immunosuppressive factor described herein is introduced into cells by lentiviral transduction.

[0290] In some embodiments, silencing of constructs encoding immunosuppressive factors results in the elimination of manipulated cells by the recipient's immune system. Furthermore, this endogenous gene Since silencing eliminates the manipulated cells, constructs containing immunosuppressive factors and inducible expression systems can be incorporated into endogenous loci to safely maintain cassette expression. In some embodiments, useful endogenous loci for incorporation are core essential loci or immune signaling factor loci. Non-limiting examples of core essential loci for such incorporation include RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn11, Psmd14, and PSMA3. Non-limiting examples of immune signaling factor loci for such integration include ligands for B2M, MIC-A / B, HLA-A, HLA-B, HLA-C, RFXANK, CTLA4, PD1, and NKG2D (e.g., MICA, MICB, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, and RAET1N / ULBP3).

[0291] In an exemplary embodiment, the conditional expression of an immunosuppressant is based on the control of the expression of the immunoregulatory factor CD47. CD47 is a component of the innate immune system that functions as a "do not eat me" signal as part of the innate immune system to block phagocytosis by macrophages. Useful immunosuppressants that can be manipulated for controlled expression include, but are not limited to, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCL21, and Mfge8.

[0292] In some embodiments, the present disclosure provides a method for producing stem cells (e.g., hypoimmunogenic pluripotent stem cells or hypoimmunogenic induced pluripotent stem cells) or differentiated cells thereof that are modified to conditionally express one of the immunosuppressive factors selected from the group consisting of CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCL21, and Mfge8. In other embodiments, the immunosuppressive factor 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, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, and HELIOS.

[0293] In some embodiments, the cells conditionally express one or more immunosuppressive factors so that, in the absence of exogenous regulatory signals, the cells are low immunogenic or have reduced low immunogenicity. In the presence of exogenous regulatory signals, the cells are recognized by immune cells and targeted for cell death or elimination. In some cases, HIP cells express immunosuppressive factors, and the function of these immunosuppressive factors allows the HIP cells to evade the immune response in the recipient. When HIP cells are exposed to exogenous regulatory signals, the expression of the immunosuppressive factor (e.g., expression at the DNA level, RNA level, or protein level) is downregulated, and therefore the HIP cells are recognized by the innate immune system in the recipient. Thus, the HIP cells undergo cell death and / or cell elimination in the recipient.

[0294] In some embodiments, the levels of immunosuppressive factors such as CD47 in manipulated cells are reduced by a DNA-level safety switch to be approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 times lower than the threshold level of expression. In some embodiments, the level of CD47 in the manipulated cells is reduced to approximately 10 to 5 times, 10 to 3 times, 9 to 1 time, 8 to 1 time, 7 to 0.5 times, 6 to 1 time, 5 to 0.5 times, 4 to 0.5 times, 3 to 0.5 times, 2 to 0.5 times, or 1 to 0.5 times lower than the threshold level of expression. In some cases, the threshold level of CD47 expression is established based on the exogenous expression of CD47 in induced pluripotent stem cells. In other cases, the threshold level of CD47 expression is established based on the expression level of CD47 in corresponding hypoimmune cells such as MHC I and MHC II knockout cells or MHC I / MHC II / TCR knockout cells.

[0295] B. Conditional upregulation methods for HIP cells and immune signaling factors Methods for the controllable expression of immune signaling factors relating to increasing the expression of these factors in order to modify the low immunogenicity of cells are described herein. Also described are HIP cells having the controllable expression of one or more immune signaling factors. In some embodiments, the immune signaling factors are selected from the group consisting of ligands for B2M, MIC-A / B, HLA-A, HLA-B, HLA-C, RFXANK, CTLA-4, PD-1, and NKG2D (e.g., MICA, MICB, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, and RAET1N / ULBP3).

[0296] Controllable expression of one or more immune signaling factors may be provided using degron-based inducible ligand stabilization systems, inducible RNA upregulation systems (e.g., inducible CRISPR activation), and inducible DNA upregulation systems. In some embodiments, the inducible DNA upregulation system includes inducible CRISPR activation (CRISPRa), a tissue-specific promoter, an inducible promoter, and a riboswitch.

[0297] A detailed description of the CRISPRa method can be found, for example, in Engreitz et al., Cold Spring Harb Perspect Biol, 2019, 11:a035386, which is incorporated herein by reference in its entirety. Detailed descriptions and examples of inductive riboswitches can be found, for example, in US9, 228, 207, US9, 993, 491, and US10, 421, 989, and in Seeliger et al., PLoS One, 2012, 7(1):e29266, which are incorporated herein by reference in their entirety.

[0298] C. Bicistronic constructs for expressing safety switches and target factors This specification describes a system for associating the expression of a safety switch in a cell with the expression of a target factor (e.g., a low-immunity factor or an essential cell factor), thereby ensuring the elimination of the cell by silencing the expression of the safety switch. By positioning the safety switch at the 5' end of the gene encoding the target factor (e.g., a low-immunity factor or an essential cell factor), a silencing event or mutation that interferes with the expression of the safety switch also interferes with the expression of the target factor (e.g., a low-immunity factor or an essential cell factor), thereby rendering the mutated cell unviable and subjecting it to apoptosis.

[0299] The primary component of the bisistronic construct of this technology is a safety switch that kills cells containing the construct in the presence of a drug or prodrug. In some embodiments, this disclosure provides low immunogenic cells (e.g., HIP stem cells or their differentiated cells) containing a “suicide gene” (or “suicide switch”). The suicide gene is incorporated to function as a “safety switch” that can cause the death of the low immunogenic cells if they were to proliferate and divide in an undesirable manner. The suicide gene removal method includes a suicide gene in a gene transfer vector that codes for a protein that results in cell killing only when activated by a specific compound. The suicide gene is used to convert a non-toxic compound into a highly toxic one. It can encode enzymes that selectively convert substances into metabolites.

[0300] Bicistronic constructs for the co-expression of a safety switch (e.g., a safety protein) and a target factor such as a low-immunity factor or an essential cell factor are provided herein. In some embodiments, the co-expression of the safety switch and the low-immunity molecule is obtained by the expression of a polycistronic transcript in which the target factor and the safety switch are separated by a ribosome skipping sequence. In some embodiments, the expression of the construct (e.g., a cassette) is controlled either by a promoter in the case of genomic location-independent transcriptional regulation, or by a splice acceptor to allow control of the payload (e.g., safety switch and target factor) by an endogenous promoter following the incorporation of the construct into a selected target gene.

[0301] In some embodiments, the safety switch transgene of the construct is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 transgene, RQR8 transgene, CD20 transgene, CCR4 transgene, CD19 transgene, MUC1 transgene, EGFR transgene, HER2 transgene, GD2 transgene, PSMA transgene, CD16 transgene, and CD30 transgene. In some embodiments, the HER1 transgene, RQR8 transgene, CD20 transgene, CCR4 transgene, HER2 transgene, CD19 transgene, MUC1 transgene, EGFR transgene, GD2 transgene, PSMA transgene, CD16 transgene, or CD30 transgene includes their epitopes. In some cases, the transgene includes a gene encoding an epitope selected from the group consisting of CD20 epitope, CCR4 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, HER2 epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.In some embodiments, the transgene is an anti-CD20 therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLib, and their biosimilars; an anti-CCR4 therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; an anti-HER2 therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and its biosimilars; an anti-CD19 therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; an anti-MUC1 therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; tomzotuximab; RO5083945 (GA201). The present invention includes an anti-EGFR therapeutic antibody selected from the group consisting of cetuximab and its biosimilars, an anti-GD2 therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLlc and its biosimilars, an anti-PSMA therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, an anti-CD30 or anti-CD16 therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or an anti-CD20 or anti-CD16 therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars, which includes an epitope that binds to the CD20 gene product and is recognized by such an antibody.

[0302] Instructions for the safety switch and its use can be found in Dusgunes, N. (2019) Origins of Suicide Gene Therapy. In: Dusgunes N. (eds) Suicide Gene Therapy. Methods in Molecular Biology, vol 1895. Humana Press, New York, NY (on HSVtk, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, and horseradish peroxidase), Zhou and Brenner, Exp Hematol, 2016, 44(11):1013-1019 (on iCaspase9), Wang et al., Blood, 20 This is described in 01,18(5),1255-1263 (on huEGFR), US20180002397 (on HER1), and Philip et al., Blood, 2014,124(8),1277-1287 (on RQR8).

[0303] In some cases, the thymidylate synthase gene or a variant thereof is included in the construct. For example, cells expressing thymidylate synthase are sensitive to certain prodrugs, including ganciclovir. Intracellular thymidylate synthase expression makes cells sensitive to the prodrug ganciclovir. In another embodiment, the CD20 gene is included. CD20-positive cells can be killed by treatment with an anti-CD20 antibody (e.g., rituximab or its biosimilars or substitutes). In some cases, the HSVtk transgene is regulated by the exogenous factor ganciclovir. In some cases, the cytosine deaminase transgene is regulated by the exogenous factor 5-fluorocytosine. In some cases, the nitroreductase transgene is regulated by the exogenous factor CB1954. In some cases, the purine nucleoside phosphorylase transgene is regulated by the exogenous factor 6-methylpurine deoxyriboside or fludarabine. In some cases, the horseradish peroxidase transgene is regulated by the exogenous factor indole-3-acetic acid.

[0304] In some cases, iCaspase9 transgenes are regulated by the exogenous factors rimiducid (AP1903), AP20187, or rapamycin. In some cases, human truncated EGFR transgenes (e.g., EGFRt) are regulated by the exogenous antibody cetuximab, or its variants that recognize the same or similar epitopes. In some cases, human HER1 transgenes are regulated by the exogenous antibody cetuximab, or its variants that recognize the same or similar epitopes. In some cases, human RQR8 transgenes are regulated by the exogenous antibody rituximab, or its variants that recognize the same or similar epitopes.

[0305] In some embodiments, the CD20 gene product is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLib, and their biosimilars; the CCR4 gene product is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 gene product is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; the CD19 gene product is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars; the MUC1 gene product is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars; and the EGFR gene product is recognized by Thomzo The GD2 gene product is recognized by a therapeutic antibody selected from the group consisting of tuximab, RO5083945 (GA201), cetuximab, and their biosimilars; the PSMA gene product is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLlc, and their biosimilars; the PSMA gene product is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars; the CD30 or CD16 gene product is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 gene product is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0306] In some embodiments, the safety switch gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In many embodiments, the inducible caspase protein is iCasp9. In some cases, iCasp9 is transmitted via a sequence of amino acids. The human FK506-binding protein contains sequence mutations FKBP12 and F36V linked to the gene encoding tocaspase 9. FKBP12-F36V binds with high affinity to the small molecule dimerizers limitid or AP1903. Therefore, the suicide function of iCasp9 is activated by administration of a dimer-inducing compound (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization triggers rapid induction of apoptosis. See, for example, WO2011146862, Stasi et al, N.Engl.J.Med 365;18 (2011), and Tey et al, Biol.Blood Marrow Transplant.13:913-924 (2007), each of which is incorporated herein by reference in whole.

[0307] In some embodiments, the safety switch transgene is an antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)-dependent safety switch. In some cases, the safety switch transgene includes an EGFR fragment or epitope, a CD20 fragment or epitope, or a CD19 fragment or epitope.

[0308] In some cases, the human EGFR safety switch is controlled by the antibody cetuximab, its variants that recognize the same or similar epitopes, or another anti-EGFR antibody. In some cases, the human CD19 safety switch is controlled by the antibody bevacizumab, its variants that recognize the same or similar epitopes, or another anti-CD19 antibody. In some cases, the human CD20 safety switch is controlled by the antibody rituximab, its variants that recognize the same or similar epitopes, or another anti-CD20 antibody.

[0309] In some embodiments, the safety switch is co-expressed with a low-immunity factor selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In a particular embodiment, the low-immunity factor is CD47.

[0310] In some cases, the bicistronic constructs also include natural or synthetic terminators. For example, those included for use in the constructs of this disclosure include any one of the terminators specified and described in Chen et al., Nature Methods, 2013, 10, 659-664, the contents of which are incorporated herein by reference. In some embodiments, the terminator is located at the 3' end of the expression construct. In some embodiments, the terminator is operably ligated to a target factor gene (e.g., a low-immunity factor gene or an essential cell factor gene).

[0311] In some cases, the bisistronic construct includes ribosome skipping sequences, such as sequences encoding IRES sequences or 2A coding sequences, but is not limited to these. Non-exclusive examples of self-cleaving 2A coding sequences include T2A, P2A, E2A, and F2A. Examples of T2A, P2A, E2A, and F2A peptide sequences are shown in Table 2.

[0312] In some cases, the bisistronic construct includes a linker positioned between the safety switch and the target factor, such as a peptide linker or a flexible linker. Examples of linkers are provided in Table 2.

[0313] In some embodiments, the bisistronic construct includes a transcriptional regulatory element. In some cases, the transcriptional regulatory element controls the expression of safety switches and low-immunity factors. In some embodiments, the transcriptional regulatory element is a promoter or a splice acceptor. In some embodiments, the promoter is a constitutive promoter selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. Examples of constitutive promoter sequences are provided in Table 4.

[0314] In some embodiments, the bisistronic construct is designed for lentiviral expression. Lentiviral vectors containing bisistronic constructs, as outlined, are provided herein. A useful lentiviral vector backbone is selected according to the cell type to be transduced.

[0315] This specification provides a bicistronic construct comprising a safety switch introduction gene operably ligated to a ribosome skipping sequence and / or a linker-coding sequence, which is operably ligated to a gene encoding a target factor (e.g., a low-immunity factor or essential cell factor). Note that, for example, by positioning the safety switch at the 5' end of the low-immunity factor gene in a bicistronic form, it is ensured that silencing events such as frameshift mutations that inactivate the safety switch also inactivate the low-immunity factor gene. In some embodiments, the expression of the bicistronic construct is controlled by a constitutive promoter operably ligated to a safety switch introduction gene operably ligated to a ribosome skipping sequence and / or a linker-coding sequence, which is operably ligated to the low-immunity factor gene. In some embodiments, the construct also comprises a polyadenylated sequence at its 3' end. In some embodiments, the construct comprises a natural or synthetic terminator.

[0316] Also provided are bicistronic constructs comprising a ribosome skipping sequence operably linked to a safety switch transgene or a low-immunity factor gene (or essential cell factor gene) operably linked to a linker. In some cases, in this bicistronic form, a frameshift mutation in the safety switch does not inactivate the low-immunity factor (or essential cell factor). In some embodiments, the expression of the bicistronic construct is controlled by a constitutive promoter operably linked to a ribosome skipping sequence operably linked to a safety switch transgene or to a target factor gene operably linked to a linker. In some embodiments, the construct also comprises a polyadenylated sequence at its 3' end. In some embodiments, the construct comprises a native or synthetic terminator.

[0317] For all of these techniques, recombinant nucleic acids as outlined herein are generated using well-known recombinant techniques. In certain embodiments, recombinant nucleic acids encoding any of the factors described herein may be operably ligated to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be appropriate for the host cell and subject being treated. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for various host cells. Typically, one or more regulatory nucleotide sequences may, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters, as known in the art, are also contemplated. The promoter may be a native promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may reside on an episome, such as a plasmid, in a cell, or the expression construct may be inserted into a chromosome. In specific embodiments, the expression vector may allow selection of transformed host cells. The expression vector includes a selectable marker gene. In certain embodiments, the expression vector includes a nucleotide sequence encoding a variant polypeptide operably linked to at least one control sequence. Control sequences for use herein include promoters, enhancers, and other expression regulatory elements. In certain embodiments, the expression vector is designed for the selection of the host cell to be transformed, a specific variant polypeptide to be expressed, the copy number of the vector, the ability to control its copy number, or any other protein encoded by the vector, such as an antibiotic marker.

[0318] Examples of suitable mammalian promoters include, for example, promoters from the following genes: the hamster ubiquitin / S27a promoter (WO97 / 15664), the monkey vacuolar virus 40 (SV40) early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rouss sarcoma virus (RSV) long-chain terminal repeat region, the mouse mammary cancer virus promoter (MMTV), the Moloney's mouse leukemia virus long-chain terminal repeat region, and the human cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters include actin, immunoglobulin, or heat shock promoters. In additional embodiments, promoters for use in mammalian cells can be obtained from the genomes of viruses such as polyomavirus, fowlpox virus (UK2,211,504, published July 5, 1989), bovine papillomavirus, arowana sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and monkey virus 40 (SV40). The early and late promoters of SV40 are conveniently obtained as SV40 restriction fragments that also contain the SV40 viral replication origin (Fiers et al, Nature 273:113-120 (1978)). The very early promoter of human cytomegalovirus is conveniently obtained as a Hind III restriction enzyme fragment (Greenaway et al, Gene 18:355-360 (1982)). The aforementioned references are incorporated in their entirety by reference.

[0319] The process for introducing the polynucleotides described herein into cells can be achieved by any preferred technique. Preferred techniques include, but are not limited to, calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. In some embodiments, the polynucleotides are introduced into cells via viral transduction (e.g., lentiviral transduction). Once modified, the presence of expression of any of the molecules described herein can be assayed using known techniques such as Western blotting, ELISA assay, FACS assay, etc.

[0320] In some embodiments, the constructs described herein are introduced into isolated cells, such as isolated mammalian cells and isolated human cells. In some embodiments, the cells are stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, or differentiated cells thereof. In some cases, the cells are hypoimmunogenic. Hypoimmunogenic cells and methods for generating such cells are described herein.

[0321] D. Homology-Directed Restoration (HDR) of Safety Switches and Target Factors In some embodiments, a construct designed for co-expression of a safety switch (e.g., degron) and a target factor (e.g., an immunosuppressive factor or essential cell factor) as a single mRNA transcript is inserted at an endogenous locus by homology-directed repair (HDR). In this configuration, the inserted element disrupts the endogenous coding sequence. In some embodiments, the endogenous locus is an essential cell factor locus. In such examples, the introduced tandem construct, expressed under the control of an endogenous promoter, compensates for the deletion of the essential cell factor gene and creates a codependency for safety switch expression. In some cases, the safety switch is inserted into the essential gene. Itch knock-in allows for avoidance of expression pressure.

[0322] In some embodiments, a construct containing a promoter and a bicistronic expression construct is introduced by HDR at a genomic locus such as a safe harbor locus, an immune signaling locus, or an essential cell factor locus, followed by knockout of both alleles of an endogenous essential cell factor gene (or, optionally, a gene encoding an immunosuppressive factor) using a targeted nuclease. In this configuration, a silent mutation in the sequence encoding the essential cell factor gene is introduced into the bicistronic construct to confer resistance to nuclease cleavage. The introduced tandem expression construct compensates for the deletion of the essential cell factor gene and creates a codependency for the expression of the safety switch.

[0323] In some embodiments, the construct for HDR into a safe harbor locus includes a first homology arm homologous to a first endogenous sequence of the safe harbor locus, a safety switch transgene, a sequence encoding a ribosome skipping sequence and / or a linker, an immunosuppressor gene (or essential cell gene), a polyadenylation sequence, and a second homology arm homologous to a second endogenous sequence of the safe harbor locus. In some embodiments, the construct for HDR into an immunosignaling locus includes a first homology arm homologous to a first endogenous sequence of the immunosignaling locus, a safety switch transgene, a sequence encoding a ribosome skipping sequence and / or a linker, an immunosuppressor gene, a polyadenylation sequence, and a second homology arm homologous to a second endogenous sequence of the immunosignaling locus. In some embodiments, the construct for HDR into an essential cell factor locus includes a first homology arm homologous to a first endogenous sequence of the essential cell factor locus, a safety switch transgene, a sequence encoding a ribosome skipping sequence and / or a linker, an immunosuppressor gene, a polyadenylation sequence, and a second homology arm homologous to a second endogenous sequence of the essential cell factor locus. In some cases, the transcriptional regulatory element is located at 5' of the safety switch transgene.

[0324] In some embodiments, the transcriptional regulatory element is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter (also known as the CAG promoter), SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0325] In some embodiments, the safety switch gene of the construct is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspase9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD16 gene, and CD30 gene.

[0326] As described above, immunosuppressive factors may be, but are not limited to, CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some cases, essential cellular factors may be, but are not limited to, RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunits, proteasome subunits, and spliceosome subunits. In some embodiments, immunosuppressive factors such as CD47, along with safety switch genes, are used to reduce the silencing of essential cellular factor genes. It is introduced into the gene to be modified. In some cases, the gene encodes an essential cellular factor selected from the group consisting of proteasome subunit Psmd14, ribosomal subunit Rps2, and ribosomal subunit RpL32.

[0327] In some embodiments, the linker is a peptide linker, a flexible linker, etc., located between the safety switch and the low-immunity factor. In some embodiments, the linker is a peptide linker, a flexible linker, etc., located between the safety switch and the essential cellular factor. Examples of linkers are provided in Table 2.

[0328] In some embodiments, the ribosome skipping sequence includes a sequence encoding an IRES sequence or a sequence encoding a 2A coding sequence. Non-limiting examples of self-cleaving 2A coding sequences include T2A, P2A, E2A, and F2A. Examples of T2A, P2A, E2A, and F2A peptide sequences are shown in Table 2.

[0329] In some embodiments relating to target incorporation, the safe harbor locus is selected from the group consisting of the AAVS1 locus, CLBYL locus, CXCR4 locus, Rosa26 locus, and CCR5 locus. In many embodiments, the safe harbor locus is either the CLBYL locus or the CCR5 locus. In some embodiments, the immune signaling locus is selected from the group consisting of ligands for B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA4, PD1, and NKG2D (e.g., MICA, MICB, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, and RAET1N / ULBP3). In some embodiments, the essential cell factor locus is selected from the group consisting of the RpS2 locus, RpS9 locus, RpS11 locus, RpS13 locus, RpS18 locus, RpL8 locus, RpL11 locus, RpL32 locus, RpL36 locus, Rpn22 locus, Psmd14 locus, PSMA3 locus, ribosomal subunit locus, proteasome subunit locus, and spliceosome subunit locus.

[0330] Targeted incorporation of safety switches and immunosuppressive factors into selected gene loci can be carried out using targeted nuclease technologies, such as the CRISPR-based and non-CRISPR-based methods described herein.

[0331] Cells expressing safety switches and immunosuppressive factors at the Safe Harbor locus are outlined herein. Cells expressing safety switches and immunosuppressive factors at immune signaling loci are also provided. Expression of safety switches is associated with the expression of immunosuppressive factors in living cells. In some embodiments, the cells are mammalian cells and isolated human cells. In some embodiments, the cells are stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, or differentiated cells thereof. In some cases, the cells are hypoimmunogenic. Hypoimmunogenic cells and methods for generating such cells are described herein.

[0332] In some embodiments, a homology-independent donor construct is provided, comprising a 5' long-terminal repeat sequence (LTR) containing a left-side element (LE), a splice acceptor-virus 2A peptide (SA-2A) element, a safety switch transgene, a sequence encoding a ribosome skipping sequence or linker, an immunosuppressor gene, a polyadenylation sequence, and a 3' LTR containing a right-side element (RE) at its 5' to 3' end. In some embodiments, the construct is introduced into cells via Cas9-induced HDR. In some embodiments, the construct is introduced into cells via homology-independent integration.

[0333] Also provided are homology-independent donor constructs comprising a 5' long-terminal repeat sequence (LTR) containing a left-side element (LE), a splice acceptor-virus 2A peptide (SA-2A) element, a low-immunity factor gene, a ribosome skipping sequence or linker encoding sequence, a safety switch introduction gene, a polyadenylation sequence, and a 3' LTR containing a right-side element (RE) at the 5' to 3' end. In some embodiments, the construct is introduced into cells via Cas9-induced HDR. In some embodiments, the construct is introduced into cells via homology-independent integration.

[0334] In some embodiments, one of the constructs described is introduced into isolated cells, such as isolated mammalian cells and isolated human cells. In some embodiments, the cells are stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, or differentiated cells thereof. In some cases, the cells are hypoimmunogenic. Hypoimmunogenic cells and methods for generating such cells are described herein.

[0335] E. Low immunofactor-safety switch fusion protein ADCC and CDC function via immune effector cells by recognizing antibodies bound to the extracellular surface of cells. ADCC / CDC are activated by the expression of antibody-recognized epitopes. Thus, this system can act as an effective safety switch. Fusion proteins comprising epitopes and hypoimmune molecules are provided herein. The fusion proteins provide a double safety mechanism against the elimination of engineered hypoimmune cells. Peptidelic epitopes such as CD20 fragments recognized by rituximab (referred to as "CD20 mimotopes") are linked to extracellular or membrane-bound hypoimmune molecules such as CD47, but are not limited to CD47.

[0336] In some embodiments, the fusion protein comprises a low-immunity factor and a peptidolytic epitope. In some embodiments, the fusion protein comprises a low-immunity factor, a peptidolytic epitope, and a linker.

[0337] In some embodiments, the fusion protein includes a low-immunity factor and a peptidic epitope from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a peptidic epitope and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a low-immunity factor, a linker, and a peptidic epitope from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a peptidic epitope, a linker, and a low-immunity factor from the N-terminus to the C-terminus.

[0338] In some embodiments, the fusion protein comprises a linker, a low-immunity factor, a linker, and a peptidolytic epitope from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises a low-immunity factor, a linker, a peptidolytic epitope, and a linker from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises a linker, a peptidolytic epitope, a linker, and a low-immunity factor, and a linker from the N-terminus to the C-terminus.

[0339] In one embodiment, the fusion protein includes a surface-exposed human CD20 epitope and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a surface-exposed human CD20 epitope, a linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a linker, a surface-exposed CD20 epitope, a linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a surface-exposed CD20 epitope, a linker, a low-immunity factor, and a linker from the N-terminus to the C-terminus. In certain embodiments, the fusion protein includes a low-immunity factor and a surface-exposed human CD20 epitope from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a surface-exposed human CD20 epitope, a linker, and a low-immunity factor from the N-terminus to the C-terminus. It contains an exposed human CD20 epitope. In some embodiments, the fusion protein includes a linker, a low-immunity factor, a linker, and a surface-exposed human CD20 epitope from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes a low-immunity factor, a linker, a surface-exposed human CD20 epitope, and a linker from the N-terminus to the C-terminus.

[0340] In some embodiments, the fusion protein includes an optional linker, a human CD20 epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human CD20 epitope is recognized by rituximab, its variant, or another anti-CD20 antibody. In some embodiments, the fusion protein includes an optional linker, a human CD19 epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human CD19 epitope is recognized by bevacizumab, its variant, or another anti-CD19 antibody. In some embodiments, the fusion protein includes an optional linker, a human EGFR epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human EGFR epitope is recognized by cetuximab, its variant, or another anti-EGFR antibody. In some embodiments, the fusion protein includes an optional linker, a human CCR4 epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human CCR4 epitope is recognized by an anti-CCR4 antibody. In some embodiments, the fusion protein includes an optional linker, a human MUC1 epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human MUC1 epitope is recognized by an anti-MUC1 antibody. In some embodiments, the fusion protein includes an optional linker, a human CD16 epitope or a human CD30 epitope, an optional linker, and a low-immunity factor from the N-terminus to the C-terminus. In some embodiments, the human CD30 epitope is recognized by an anti-CD30 antibody or its bispecific antibody. In some embodiments, the human CD16 epitope is recognized by an anti-CD16 antibody or its bispecific antibody. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an optional linker, a human CD20 epitope or a human CD16 epitope, an optional linker, and a low-immunity factor. In some embodiments, the human CD20 epitope is recognized by an anti-CD20 antibody or a bispecific antibody thereof.In some embodiments, the human CD16 epitope is recognized by an anti-CD16 antibody or a bispecific antibody thereof. In some embodiments, the fusion protein includes, from N-terminus to C-terminus, an optional linker, a human PSMA epitope, an optional linker, and a low-immunity factor. In some embodiments, the human PSMA epitope is recognized by an anti-PSMA antibody. In some embodiments, the fusion protein includes, from N-terminus to C-terminus, an optional linker, a human GD2 epitope, an optional linker, and a low-immunity factor. In some embodiments, the human GD2 epitope is recognized by an anti-GD2 antibody. In other embodiments, the order of the peptide epitope and the low-immunity factor is reversed.

[0341] In other embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a human CD47 fragment containing the IgV domain of CD47, a linker, a peptidolytic epitope, another linker, and a human CD47 transmembrane domain.

[0342] In another embodiment, a bisistronic construct is provided herein that comprises a transcriptional regulatory element, a sequence encoding a peptidolytic epitope, a ribosome skipping sequence, and a sequence encoding a low-immunity factor at its 5' to 3' end. In some embodiments, the peptidolytic epitope is selected from the group consisting of CD20 epitope, CCR4 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, HER2 epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

[0343] In some embodiments, the low-immunity factors are CD47, CD24, CD200, HLA-G, The group consists of HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms. In certain embodiments, the low immunofactor is CD47. In some embodiments, the linker is selected from one of those listed in Table 2.

[0344] In some embodiments, one of the peptide epitopes is selected from the group consisting of CD20 epitope, CCR4 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, HER2 epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

[0345] In some embodiments, the CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. In some embodiments, the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars. In some embodiments, the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars. In some embodiments, the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars. In some embodiments, the MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars. In some embodiments, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars. In some embodiments, the GD2 epitope is recognized by a therapeutic antibody selected from the group consisting of Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c.60C3-RLIc, and their biosimilars. In some embodiments, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars. In some embodiments, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars. In some embodiments, the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

[0346] Any one of the constructs described may be introduced into isolated cells, such as isolated mammalian cells and isolated human cells. In some embodiments, the cells are stem cells, embryonic stem cells, pluripotent stem cells, adult stem cells, or differentiated cells thereof. In some cases, the cells are hypoimmunogenic. Hypoimmunogenic cells and methods for generating such cells are described herein.

[0347] F. Conditional HIP cells with modified expression of MHC I, MHC II, and TCR complexes In one embodiment, the techniques disclosed herein relate to the use of safety switches to control the expression of target factors in pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells), and primary T cells. In certain embodiments, pluripotent stem cells, differentiated cells derived therefrom, and primary T cells are manipulated toward reduced or deleted expression of MHC class I and MHC class II human leukocyte antigens. In certain embodiments, pluripotent stem cells, differentiated cells derived therefrom, and primary T cells are manipulated toward reduced or deleted expression of MHC class I and MHC class II human leukocyte antigens, as well as reduced or deleted expression of one or more T cell receptor (TCR) complexes. In some cases, the deletion or reduction of expression of MHC class I antigens, MHC class II antigens, and / or one or more TCR complexes is achieved using an inducible gene modification system.

[0348] In some embodiments, the disclosure provides pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells), primary T cells, and populations thereof, comprising a genome in which genes have been edited to delete a continuous sequence of genomic DNA, thereby reducing or eliminating the surface expression of MHC class I molecules in the cell or population. In certain embodiments, the disclosure provides pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells), primary T cells, and populations thereof, comprising a genome in which genes have been edited to delete a continuous sequence of genomic DNA, thereby reducing or eliminating the surface expression of MHC class II molecules in the cell or population. In certain embodiments, the disclosure provides pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells), primary T cells, and populations thereof, comprising a genome in which one or more genes have been edited to delete a continuous sequence of genomic DNA, thereby reducing or eliminating the surface expression of one or more TCR complexes in the cell or population. In further embodiments, the disclosure provides pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells), primary T cells, and populations thereof, comprising a genome in which one or more genes have been edited to delete a continuous sequence of genomic DNA, thereby reducing or eliminating the surface expression of one or more TCR complexes in the cell or population.

[0349] In some embodiments, the cells include genomic modifications of one or more target polynucleotide sequences that control the expression of MHC I and / or MHC II. In some embodiments, one or more target polynucleotide sequences are modified using a gene editing system. In some embodiments, the target polynucleotide sequences are one or more selected from the group consisting of B2M, CIITA, and NLRC5. In certain embodiments, the cell genome is modified to reduce or delete essential components of HLA expression. In additional embodiments, the cells include genomic modifications of one or more target polynucleotide sequences that control the expression of one or more TCR complexes. In some embodiments, one or more target polynucleotide sequences are modified using a gene editing system. In some embodiments, the target polynucleotide sequences are one or more selected from the group consisting of TRAC and TRB.

[0350] In some embodiments, the cells and methods described herein include genome editing of human cells to cleave the CIITA gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences, including but not limited to B2M, NLRC5, TRAC, and TRB. In some embodiments, the cells and methods described herein include genome editing of human cells to cleave the B2M gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences, including but not limited to CIITA, NLRC5, TRAC, and TRB. In some embodiments, the cells and methods described herein include genome editing of human cells to cleave the NLRC5 gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences, including but not limited to B2M, CIITA, TRAC, and TRB. In some embodiments, the cells and methods described herein include genome editing of human cells to cleave the TRAC gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences, including but not limited to B2M, CIITA, NLRC5, and TRB. In some embodiments, the cells and methods described herein involve genome editing of human cells to cleave the TRB gene sequence, Furthermore, this includes editing the genome of such cells to modify one or more additional target polynucleotide sequences, such as B2M, CIITA, NLRC5, and TRAC, but not limited to these.

[0351] In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise genomic modification of the B2M gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise genomic modification of the CIITA gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise genomic modification of the TRAC gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise genomic modification of the TRB gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise genomic modification of B2M and CIITA. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, and TRAC genes. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, and TRB genes. In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, and primary T cells comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the cell is a B2M - / - , CIITA - / - cell. In many embodiments, the cell is a B2M - / - , CIITA - / - , TRAC - / - cell. In many embodiments, the cell is a B2M - / - , CIITA - / - , TRB - / - cell. In some embodiments, the cell is a B2M インデル / インデル , CIITA インデル / インデル cell. In some embodiments, the cell is a B2M インデル / インデル , CIITA インデル / インデル , TRAC インデル / インデル cell. In some embodiments, the cell is a B2M インデル / インデル , CIITA インデル / インデル , TRB インデル / インデル cell. In some embodiments, the cell is a B2M インデル / インデル, CIITA インデル / インデル , TRAC インデル / インデル TRB インデル / インデル These are cells. In some embodiments, the modified cells described are pluripotent stem cells, induced pluripotent stem cells, cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, unregulated T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memory T cells, native memory T cells, memory stem cells (Tse), and γδ This includes T cells and any other subtypes of T cells.

[0352] In some embodiments, one or more genes selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR are inactivated in the cells. These genes may be inactivated using homology-dependent repair or site-specific nucleases. In some embodiments, one or both alleles of these genes are inactivated.

[0353] 1. CIITA In certain embodiments, the technology modulates the expression of MHC II genes (e.g., by reducing or eliminating) by targeting and regulating (e.g., reducing or eliminating) the expression of class II transactivators (CIITA). In some embodiments, the regulation occurs using the CRISPR / Cas system. CIITA is a nucleotide polymorphism (LR), or nucleotide polymorphism (nucleotide polymorphism). The binding domain (NBD) is a member of the leucine-rich repeat sequence (LRR) family of proteins that regulates MHC II transcription by associating with the MHC enhanceosome.

[0354] In some embodiments, the target polynucleotide sequence of the technology 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.

[0355] In some embodiments, reducing or eliminating CIITA expression reduces or eliminates the expression of one or more of the following MHC class II cells: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0356] In some embodiments, the cells described herein include gene modifications at the locus encoding the CIITA protein. In other words, the cells include gene modifications at the CIITA locus. In some cases, the nucleotide sequence encoding the CIITA protein is defined in reference number NM_000246.4 and NCBI Genbank number U18259. In some cases, the CIITA locus is described in NCBI gene ID number 4261. In a particular example, the amino acid sequence of CIITA is shown as NCBI GenBank number AAA88861.1. Further descriptions of the CIITA protein and locus can be found in Uniprot number P33076, HGNC reference number 7067, and OMIM reference number 600005.

[0357] In some embodiments, the low immunogenic cells outlined herein include gene modifications targeting the CIITA gene. In some embodiments, the gene modification targeting the CIITA gene by a rare-cutting endonuclease includes 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, 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 in Table 12 of WO2016183041, which is incorporated herein by reference. In some embodiments, the cells have a reduced ability to induce an immune response in the recipient.

[0358] Assays for testing whether the CIITA gene is inactivated are known and described herein. In one embodiment, the resulting PCR-mediated gene modification of the CIITA gene and reduction in HLA-II expression can be assayed by FACS analysis. In another embodiment, CIITA protein expression is detected using Western blotting of cell lysates probed with an antibody against the CIITA protein. In yet another embodiment, the presence of inactivating gene modifications is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0359] 2. B2M In certain embodiments, the techniques disclosed herein modulate (e.g., reduce or eliminate) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of accessory chain B2M. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) the expression of B2M, surface transport of MHC-I molecules is blocked, making cells less immunogenic. In some embodiments, the cells have a reduced ability to induce an immune response in the recipient.

[0360] In some embodiments, the target polynucleotide sequence of this technology 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.

[0361] In some embodiments, the reduction or elimination of B2M expression reduces or eliminates the expression of one or more of the following MHC I molecules, namely HLA-A, HLA-B, and HLA-C.

[0362] In some embodiments, the cells described herein include gene modifications at the locus encoding the B2M protein. In other words, the cells include gene modifications at the B2M locus. In some cases, the nucleotide sequence encoding the B2M protein is defined in reference number NM_004048.4 and Genbank number AB021288.1. In some cases, the B2M locus is described in NCBI gene ID number 567. In a particular example, the amino acid sequence of B2M is shown as NCBI GenBank number BAA35182.1. Further descriptions of the B2M protein and locus can be found in Uniprot number P61769, HGNC reference number 914, and OMIM reference number 109700.

[0363] In some embodiments, the low immunogenic cells outlined herein include gene modifications targeting the B2M gene. In some embodiments, the gene modification targeting the B2M gene by a rarecut endonuclease includes a Cas protein or a polynucleotide encoding a 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. 81240-85644 in Table 15 of WO2016183041, which is incorporated herein by reference.

[0364] Assays for testing whether the B2M gene is inactivated are known and described herein. In one embodiment, the resulting PCR-mediated gene modification of the B2M gene and reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, B2M protein expression is detected using Western blotting of cell lysates probed with an antibody against the B2M protein. In yet another embodiment, the presence of inactivating gene modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0365] 3.NLRC5 In certain embodiments, the technology modulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting and regulating (e.g., reducing or eliminating) the expression of the NLR family, CARD domain-containing 5 / NOD27 / CLR16.1 (NLRC5). In some embodiments, the regulation occurs using the CRISPR / Cas system. NLRC5 is an essential regulator of the MHC-I mediated immune response, and like CIITA, NLRC5 is highly induceable by IFN-γ and can translocate into the nucleus. NLRC5 activates the promoters of MHC-I genes, inducing the transcription of MHC-I and related genes involved in MHC-I antigen presentation.

[0366] In some embodiments, the target polynucleotide sequence of this technology 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.

[0367] In some embodiments, reducing or eliminating NLRC5 expression reduces or eliminates the expression of one or more of the following MHC I molecules, namely HLA-A, HLA-B, and HLA-C.

[0368] In some embodiments, the low immunogenic cells outlined herein include gene modifications targeting the NLRC5 gene. In some embodiments, the gene modification targeting the NLRC5 gene by a rarecut endonuclease includes a Cas protein or a polynucleotide encoding a 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 (Table 14 of WO2016183041) provided herein. In some embodiments, the cells have a reduced ability to induce an immune response in the recipient.

[0369] Assays for testing whether the NLRC5 gene is inactivated are known and described herein. In one embodiment, the resulting PCR-mediated gene modification of the NLRC5 gene and reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, NLRC5 protein expression is detected using Western blotting of cell lysates probed with an antibody against the NLRC5 protein. In yet another embodiment, the presence of inactivating gene modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0370] 4. TRAC In certain embodiments, the techniques disclosed herein modulate (e.g., reduce or eliminate) the expression of TCR genes, including the TRAC gene, by targeting and modulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor alpha chain. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) TRAC expression, surface transport of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce an immune response in the recipient.

[0371] In some embodiments, the target polynucleotide sequence of the Technology is a variant of TRAC. In some embodiments, the target polynucleotide sequence is a homolog of TRAC. In some embodiments, the target polynucleotide sequence is an orthologue of TRAC.

[0372] In some embodiments, reducing or eliminating TRAC expression reduces or eliminates TCR surface expression.

[0373] In some embodiments, the cells described herein include gene modifications at the locus encoding the TRAC protein. In other words, the cells include gene modifications at the TRAC locus. In some cases, the nucleotide sequence encoding the TRAC protein is defined in Genbank number X02592.1. In some cases, the TRAC locus is described in reference sequence number NG_001332.3 and NCBI gene ID number 28755. In a particular example, the amino acid sequence of TRAC is shown as Uniprot number P01848. Further descriptions of the TRAC protein and locus can be found in Uniprot number P01848, HGNC reference number 12029, and OMIM reference number 186880.

[0374] In some embodiments, the low immunogenicity cells outlined herein include gene modifications targeting the TRAC gene. In some embodiments, by rarecut endonuclease Gene modifications targeting the TRAC gene include a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOs. 532-609 and 9102-9797 of US20160348073, which is incorporated herein by reference.

[0375] Assays for testing whether the TRAC gene is inactivated are known and described herein. In one embodiment, the resulting PCR-mediated gene modification of the TRAC gene and reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRAC protein expression is detected using Western blotting of cell lysates probed with an antibody against the TRAC protein. In yet another embodiment, the presence of inactivating gene modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0376] 5. TRB In certain embodiments, the techniques disclosed herein regulate the expression of TCR genes, including the gene encoding the T cell antigen receptor beta chain (e.g., TRB or TCRB gene), by targeting and regulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor beta chain. In some embodiments, the regulation occurs using a CRISPR / Cas system. By regulating (e.g., reducing or deleting) TRB expression, surface transport of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce an immune response in the recipient.

[0377] In some embodiments, the target polynucleotide sequence of the technology is a variant of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an orthologue of TRB.

[0378] In some embodiments, reducing or eliminating TRB expression reduces or eliminates TCR surface expression.

[0379] In some embodiments, the cells described herein include gene modifications at the locus encoding the TRB protein. In other words, the cells include gene modifications at the TRB locus. In some cases, the nucleotide sequence encoding the TRB protein is defined in UniProt number P0DSE2. In some cases, the TRB locus is described in reference sequence number NG_001333.2 and NCBI gene ID number 6957. In a particular example, the amino acid sequence of TRB is shown as Uniprot number P01848. Additional descriptions of the TRB protein and locus can be found in GenBank number L36092.2, Uniprot number P0DSE2, and HGNC reference number 12155.

[0380] In some embodiments, the low immunogenic cells outlined herein include gene modifications targeting the TRB gene. In some embodiments, the gene modification targeting the TRB gene by a rarecut endonuclease includes a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOs. 610-765 and 9798-10532 of US20160348073, which is incorporated herein by reference.

[0381] Assays for testing whether the TRB gene is inactivated are known and described herein. In one embodiment, the resulting PCR-mediated gene modification of the TRB gene and reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRB protein expression is detected using Western blotting of cell lysates probed with an antibody against the TRB protein. In yet another embodiment, the presence of inactivating gene modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0382] Methods to reduce or eliminate G.MHC class I, MHC class II, and / or TCR expression. Methods for modifying or manipulating cells to reduce the expression of MHC I antigens, MHC II antigens, and / or one or more TCR complexes are provided herein. Reduction of MHC I and / or MHC II expression can be carried out, for example, by one or more of the following: (1) directly targeting polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes; (2) blocking the surface transport of all MHC-I molecules by removing B2M; (3) blocking the surface transport of all MHC-II molecules by removing CIITA; and / or (4) deletion of components of the MHC enhanceosome that are essential for HLA expression, such as LRC5, RFX-5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA.

[0383] In some embodiments, HLA expression is inhibited by targeting individual HLAs (e.g., knocking out the expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), targeting transcriptional regulators of HLA expression (e.g., knocking out the expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and / or IRF-1), blocking the surface transport of MHC class I molecules (e.g., knocking out the expression of B2M and / or TAP1), and / or targeting with HLA-Razor (see, for example, WO2016183041).

[0384] In certain embodiments, the cells disclosed herein, including but not limited to pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from such stem cells, and primary T cells, are characterized by not expressing one or more human leukocyte antigens corresponding to MHC-I and / or MHC-II (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), and therefore being low immunogenic. For example, in certain embodiments, the disclosed pluripotent stem cells and induced pluripotent stem cells are modified such that the stem cells or differentiated stem cells prepared therefrom do not express one or more of the following MHC-I molecules, namely HLA-A, HLA-B, and HLA-C, or exhibit reduced expression thereof. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C can be “knocked out” from the cells. Cells in which the HLA-A gene, HLA-B gene, and / or HLA-C gene have been knocked out may show reduced or eliminated expression of each knocked-out gene.

[0385] In certain embodiments, a gRNA that enables the simultaneous deletion of all MHC class I alleles by targeting a conserved region in an HLA gene is identified as an HLA Razor. In some embodiments, the gRNA is part of the CRISPR system. In alternative embodiments, the gRNA is part of the TALEN system. In one embodiment, an HLA Razor targeting a specific conserved region in HLA is described in WO2016183041. In other embodiments, multiple HLA Razors targeting a specific conserved region are utilized. Generally, any guide that targets a conserved region in HLA is an HLA Razor. It is understood that it can function as an azor.

[0386] The methods provided below are useful for inactivating or removing MHC class I expression, MHC class II expression, and / or TCR expression in cells such as pluripotent stem cells, differentiated cells thereof, and primary T cells, but are not limited to these. In some embodiments, genome editing techniques utilizing rare-cut endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are used to reduce or eliminate the expression of essential immunogenes in human stem cells (e.g., by deleting the genomic DNA of essential immunogenes). In certain embodiments, resistance-inducing factors are inserted into human cells using genome editing techniques or other gene regulation techniques to make them and differentiated cells prepared therefrom hypoimmunogenic. Thus, hypoimmunogenic cells have reduced or eliminated MHC I and MHC II expression. In some embodiments, these cells are nonimmunogenic in the recipient (e.g., do not induce an immune response).

[0387] Genome editing techniques enable double-strand DNA breaks at desired loci. These controlled double-strand breaks promote homologous recombination at specific loci. This process focuses on targeting specific sequences in nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequence, inducing double-strand breaks in the nucleic acid molecule. The double-strand breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0388] The implementation of a particular embodiment will, unless otherwise indicated, utilize conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the scope of the skill of those skilled in the art, many of which are described below for illustrative purposes. Such techniques are fully described in the literature.For example, 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 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 to Molecular Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q.E. Coligan, A M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances. Please refer to research papers in publications such as *in Immunology*.

[0389] In some embodiments, the rarecut endonuclease is introduced into cells containing a target polynucleotide sequence in the form of a nucleic acid encoding the rarecut endonuclease. The process of introducing the nucleic acid into cells can be achieved by any preferred technique. Preferred techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. 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.

[0390] This disclosure aims to modify a target polynucleotide sequence in any manner available to those skilled in the art using a CRISPR / Cas system. Any CRISPR / Cas system capable of modifying a target polynucleotide sequence within a cell may be used. Such a CRISPR-Cas system can utilize a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;1(6)e60). The molecular mechanisms of such Cas proteins that enable a CRISPR / Cas system to modify a target polynucleotide sequence within a cell include 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 V system.

[0391] The CRISPR / Cas system described herein can be used to modify any target polynucleotide sequence within a cell. Those skilled in the art will readily understand that a target polynucleotide sequence desirable for modification in any particular cell may correspond to any genomic sequence whose expression is associated with impairment or otherwise facilitates the entry of pathogens into the cell. For example, a target polynucleotide sequence desirable for modification in a cell may correspond to a polynucleotide sequence containing a disease-related single polynucleotide polymorphism. In such an example, the CRISPR / Cas system can be used to correct the disease-related SNP in the cell by replacing it with a wild-type allele. In another example, a polynucleotide sequence of a target gene that causes the entry or proliferation of a pathogen into a cell may be a suitable target for deletion or insertion to disrupt the function of the target gene and prevent the pathogen from entering or proliferating within the cell.

[0392] In some embodiments, the target polynucleotide sequence is a genome sequence. In some embodiments, the target polynucleotide sequence is a human genome sequence. In some embodiments, the target polynucleotide sequence is a mammalian genome sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genome sequence.

[0393] In some embodiments, the CRISPR / Cas system comprises a Cas protein and at least one or two ribonucleic acids capable of leading the Cas protein to a target motif of a target polynucleotide sequence and hybridizing thereto. As used herein, “protein” and “polypeptide” are used interchangeably to refer to a sequence of amino acid residues joined by peptide bonds (i.e., polymers of amino acids) and include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. Examples of polypeptides or proteins include gene products, native proteins, homologs, This includes paralogs, fragments, and other equivalents, variants, and analogues of the above.

[0394] In some embodiments, the Cas protein includes one or more amino acid substitutions or modifications. In some embodiments, one or more amino acid substitutions include conservative amino acid substitutions. In some cases, substitutions and / or modifications can inhibit or reduce proteolysis in cells and / or extend the half-life of the polypeptide. In some embodiments, the Cas protein may include peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may include native amino acids. In some embodiments, the Cas protein may include alternative amino acids (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may include modifications such as PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.

[0395] In some embodiments, the Cas protein includes a core Cas protein. Examples of 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 includes a Cas protein of the E. coli subtype (also known as CASS2). Examples of 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 includes a Cas protein of the Ypest subtype (also known as CASS3). Examples of Cas proteins of the Ypest subtype include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein includes a Cas protein of the Nmeni subtype (also known as CASS4). Examples of Cas proteins of the Nmeni subtype include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein includes Cas proteins of the Dvulg subtype (also known as CASS1). Examples of Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein includes Cas proteins of the Tneap subtype (also known as CASS7). Examples of Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein includes Cas proteins of the Hmari subtype. Examples of Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein includes Cas proteins of the Apern subtype (also known as CASS5). Examples of 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 includes a Cas protein of the Mtube subtype (also known as CASS6).Examples of Mtube subtype Cas proteins include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein includes RAMP module Cas proteins. Examples of RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0396] In some embodiments, the Cas protein comprises one of the Cas proteins described herein or a functional portion thereof. As used herein, “functional portion” or “functional fragment” refers to a portion of a peptide or protein factor that, when complexed with at least one ribonucleic acid (e.g., guide RNA (gRNA)), retains its ability to cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a DNA fragment. The functional portion includes a combination of functional domains of an operably linked Cas9 protein, selected from the group consisting of a binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion includes a combination of functional domains of an operably linked Cas12a protein, 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 includes a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein includes a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of the Cas12a protein includes a functional portion of a RuvC-like domain.

[0397] In some embodiments, the exogenous Cas protein may be introduced into cells in polypeptide form. In certain embodiments, the Cas protein may be conjugated or fused to a cell-permeable polypeptide or cell-permeable peptide. As used herein, “cell-permeable polypeptide” and “cell-permeable peptide” refer, respectively, to polypeptides or peptides that facilitate the uptake of molecules into cells. The cell-permeable polypeptide may contain a detectable label.

[0398] In certain embodiments, the Cas protein may be conjugated or fused to a charged protein (e.g., possessing a positive charge, a negative charge, or an overall neutral charge). Such ligation may be covalent. In some embodiments, the Cas protein may be fused to positively superpositively charged GFP to significantly increase the Cas protein's ability to permeate cells (Cronican et al. ACS Chem Biol. 2010;5(8):747-52). In certain embodiments, the Cas protein may be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Examples of PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-permeable 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 includes a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein includes a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein includes a Cas9 polypeptide fused to a positively superpositively charged GFP. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to a cell-permeable peptide. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to a penetratin domain. In some embodiments, the Cas12a protein includes a Cas12a polypeptide fused to a positively superpositively charged GFP.

[0399] In some embodiments, the Cas protein may be introduced into cells containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The process of introducing the nucleic acid into cells may be achieved by any preferred technique. Preferred techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. 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 is as described herein. It contains modified mRNA such as that which is listed.

[0400] In some embodiments, the Cas protein is complexed with one or 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 as described herein (e.g., synthetically modified mRNA).

[0401] The method of this technology intends to use any ribonucleic acid capable of guiding a Cas protein to a target motif of a target polynucleotide sequence and hybridizing to it. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that guides the Cas protein to a target motif of a target polynucleotide sequence in the cell and hybridizes to it. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that guides the Cas protein to a target motif of a target polynucleotide sequence in the cell and hybridizes to it. In some embodiments, both of one or two ribonucleic acids comprise a guide RNA that guides the Cas protein to a target motif of a target polynucleotide sequence in the cell and hybridizes to it. As will be understood by those skilled in the art, the ribonucleic acids of this technology may be selected to hybridize to a variety of different target motifs depending on the specific CRISPR / Cas system used and the sequence of the target polynucleotide. One or two ribonucleic acids may also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, one or 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, one or 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, one or two ribonucleic acids are designed to hybridize to a target motif directly adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one or two ribonucleic acids is designed to hybridize to a target motif directly adjacent to a deoxyribonucleic acid motif recognized by the Cas protein, located on both sides of a mutant allele located between the target motifs.

[0402] In some embodiments, each of one or two ribonucleic acids contains a guide RNA that directs the Cas protein to a target motif of a target polynucleotide sequence within the cell and hybridizes to it.

[0403] In some embodiments, one or two ribonucleic acids (e.g., guide RNA) are complementary to and / or hybridize with a sequence on the same strand of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNA) are complementary to and / or hybridize with a sequence on the opposite strand of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNA) are not complementary to and / or hybridize with a sequence on the opposite strand of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNA) are complementary to and / or hybridize with an overlapping target motif of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNA) are complementary to and / or hybridize with an offset target motif of the target polynucleotide sequence.

[0404] In some embodiments, nucleic acids encoding the Cas protein and at least one to two ri The nucleic acid encoding the ribonucleic acid is introduced into the cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with one or 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.

[0405] Table 1 provides examples of gRNA sequences that are useful for CRISPR / Cas-based targeting of the genes described herein. These sequences can be found in WO2016 / 183041, filed 9 May 2016, and US2016 / 0348073, filed 28 March 2016, and the disclosures of those documents, including tables, appendices, and sequence listings, are incorporated herein by reference in their entirety. [Table 1]

[0406] In some embodiments, the cells are modified using non-CRISPR-based methods. In embodiments of this invention, such methods include, but are not limited to, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing endonucleases, sequence-specific endonucleases, meganucleases, RNA silencing or RNA interference, and RNA-inducible transposases.

[0407] A "TALE-nuclease" (TALEN) is intended to be a fusion protein comprising 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 an endonuclease domain, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain may be fused to a meganuclease, such as I-CreI and I-Onul or their functional variants. In more preferred embodiments, 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 a fusion of an engineered TAL repeat sequence and the catalytic domain of l-TevI, as described in WO2012138927. The transcription activator-like effector (TALE) is a protein derived from the bacterial species Xanthomonas, containing multiple repeat sequences, each repeat sequence containing two residues (RVD) specific to each nucleotide base of the nucleic acid target sequence at positions 12 and 13. A binding domain (MBBBD) with similar modular base-per-base nucleic acid binding properties may also be derived from a novel modular protein in a different bacterial species recently discovered by the applicant. This novel modular protein has the advantage of exhibiting greater sequence variability than the TAL repeat sequence. Preferably, the RVDs associated with 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, essential amino acids 12 and 13 can be mutated toward other amino acid residues to modulate their specificity toward nucleotides A, T, C, and G, and in particular to enhance this specificity. TALEN kits are commercially available.

[0408] In some embodiments, the cells are manipulated using zinc finger nucleases (ZFNs). A “zinc finger-binding protein” is a protein or polypeptide that binds to DNA, RNA, and / or other proteins, preferably in a sequence-specific manner, as a result of the 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.” A ZFP has at least one finger, typically two, three, or six fingers. Each finger binds to 2 to 4 base pairs of DNA, typically 3 to 4 base pairs of DNA. ZFPs bind to nucleic acid sequences called target sites or target segments. Each finger typically contains a zinc-chelated DNA-binding subdomain of approximately 30 amino acids. Studies have demonstrated that a single zinc finger in this class consists of an alpha-helix containing two invariant histidine residues coordinated to zinc, along with two cysteine ​​residues in a single beta-turn (see, for example, Berg & Shi, Science 271:1081-1085 (1996)).

[0409] In some embodiments, the cells of this disclosure are prepared using homing endonucleases, which are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and induce single-strand or double-strand breaks. Homing endonucleases are highly specific and recognize DNA target sites in the range of 12–45 base pairs (bp), typically 14–40 bp. The homing endonucleases of this disclosure may correspond, for example, to LAGLIDADG homing endonuclease, HNH endonuclease, or GIY-YIG endonuclease. In some cases, the homing endonuclease is an I-CreI variant.

[0410] In some embodiments, the cells outlined herein are prepared using meganucleases. Meganucleases are, by definition, sequence-specific endonucleases that recognize large sequences (Chevalier, B.Sand B.L.Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). These can cleave specific sites in living cells, thereby enhancing gene targeting near the cleavage site by more than 1000 times (Puchta et al., Nucleic Acids Res., 1993, 21, 5034-5040, Rouet et al., Mol.Cell.Biol., 1994, 14, 8096-8106, Choulika et al., Mol.Cell.Biol., 1995, 15, 1968-1973, Puchta et 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).

[0411] In some embodiments, the described cells are prepared using RNA silencing or RNA interference (RNAi) to knock down (e.g., reduce, eliminate, or inhibit) the expression of polypeptides such as tolerogenic factors. Useful RNAi methods include those utilizing synthetic RNAi molecules, small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs), and other transient knockdown methods recognized by those skilled in the art. Reagents for RNAi, including sequence-specific shRNAs, siRNAs, and miRNAs, are commercially available. For example, in pluripotent stem cells, CIITA siRNA may be introduced into the cells or CIITA CIITA can be knocked down by transduction with an shRNA-expressing virus. 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.

[0412] In some embodiments, RNA-inducible transposases are used to integrate DNA into the genome of the cells described herein. Detailed descriptions of useful RNA-inducible transposases and their uses can be found, for example, in Klompe et al., Nature 571, 219-225 (2019) and Strecker et al., Science. Disclosed in 365, 48-53 (2019), the contents of which are incorporated herein by reference.

[0413] H. Generation of hypoimmunogenic pluripotent stem cells This disclosure provides a method for producing low immunogenic pluripotent cells. In some embodiments, the method includes generating pluripotent stem cells. The generation of mouse and human pluripotent stem cells (collectively referred to as iPSCs; miPSCs in the case of mouse cells, or hiPSCs in the case of human cells) is This is generally known in the art. As will be understood by those skilled in the art, there are various different methods for generating iPSCs. The original induction was performed from mouse embryonic or adult fibroblasts using viral introduction of four transcription factors: Oct3 / 4, Sox2, c-Myc, and Klf4. See Takahashi and Yamanaka Cell 126:663-676 (2006) (the entire work is incorporated herein by reference, in particular with respect to the techniques outlined therein). Since then, several methods have been developed. For an overview, please refer to Seki et 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 these references are explicitly incorporated herein by reference, particularly with regard to methods for generating hiPSCs (see, for example, Chapter 3 of the latter reference).

[0414] Generally, iPSCs are generated by the transient expression of one or more reprogramming factors in host cells, usually introduced using episomal vectors. Under these conditions, a small number of cells are induced to become iPSCs (this step is generally inefficient, so selection markers are not used). Once the cells are "reprogrammed" and become pluripotent, they lose the episomal vector(s) and produce the factor using endogenous genes.

[0415] As will also be understood by those skilled in the art, the number of reprogramming factors that can or may be used can vary. Generally, when fewer reprogramming factors are used, the efficiency of the transformation of cells into a pluripotent state, as well as "pluripotency," decreases. For example, fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into a smaller number of cell types.

[0416] 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, i.e., SOX2, OCT4(POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen, may be used. Generally, these reprogramming factor genes are provided on episomal vectors that are known in the art and commercially available.

[0417] Generally, as is known in the art, iPSCs are produced from non-pluripotent cells such as blood cells and fibroblasts, but not limited to these, by transiently expressing reprogramming factors as described herein.

[0418] I. Assays concerning low immunogenicity phenotype and retention of pluripotency Once low immunogenic cells are generated, they may be assayed for retention of their low immunogenicity and / or pluripotency, as described in WO2016183041 and WO2018132783.

[0419] In some embodiments, low immunogenicity is assayed using several techniques, as exemplified in Figures 13 and 15 of WO2018132783. These techniques include transplantation into allogeneic hosts and monitoring for the proliferation of low immunogenic pluripotent cells (e.g., teratomas) that evade the host immune system. In some cases, derivatives of low immunogenic pluripotent cells are used. Cells can be transduced to express cyferase and then tracked using bioluminescence imaging. Similarly, the T cell and / or B cell response of the host animal to such cells is tested to confirm that the cells do not evoke an immune response in the host animal. T cell function is assessed by ELISpot, ELISA, FACS, PCR, or mass cytometry (CYTOF). B cell response or antibody response is assessed using FACS or Luminex. As an addition or alternative, cells may be assayed for their ability to evade innate immune responses, e.g., killing by NK cells, as commonly shown in Figures 14 and 15 of WO2018132783.

[0420] In some embodiments, the immunogenicity of cells is evaluated using T cell immunoassays such as T cell proliferation assays, T cell activation assays, and T cell killing assays, which are recognized by those skilled in the art. In some cases, a T cell proliferation assay includes pre-treating cells with interferon-gamma, co-culturing the cells with labeled T cells, and assaying the presence of a T cell population (or a proliferating T cell population) after a pre-selected period of time. In some cases, a T cell activation assay includes co-culturing T cells with cells outlined herein and determining the expression levels of T cell activation markers in the T cells.

[0421] In vivo assays can be performed to assess the immunogenicity of the cells outlined herein. In some embodiments, the viability and immunogenicity of low immunogenic cells are determined using an allogeneic humanized immunodeficiency mouse model. In some cases, low immunogenic pluripotent stem cells are transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell viability, and teratoma formation. In some cases, the transplanted low immunogenic pluripotent stem cells or their differentiated cells exhibit long-term survival in the mouse model.

[0422] Additional techniques for determining the immunogenicity of cells, including low immunogenicity, are described, for example, Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, and these disclosures, including figures, figure legends, and method descriptions, are incorporated herein by reference in their entirety.

[0423] Similarly, the retention of pluripotency is tested in several ways. In one embodiment, pluripotency is assayed by the expression of certain pluripotency-specific factors, as generally described herein and shown in Figure 29 of WO2018132783. Additionally or alternatively, pluripotent cells are differentiated into one or more cell types as indicators of pluripotency.

[0424] As those skilled in the art will understand, the reduction of MHC I function (or HLA I if the cells are derived from human cells) in pluripotent cells is known in the art and can be measured using techniques such as those described below, for example, FACS techniques using labeled antibodies that bind to the HLA complex, for example, commercially available HLA-A, B, and C antibodies that bind to the alpha chain of human major histocompatibility HLA class I antigen.

[0425] In addition, cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This may be assayed by FACS analysis using antibodies against one or more components of the HLA cell surface, as discussed above.

[0426] The 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, FACS, and RT-PCR, using antibodies against the protein in question.

[0427] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is performed as is known in the art (e.g., Figure 21 of WO2018132783), and is generally performed using either Western blotting or FACS analysis based on commercially available antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens.

[0428] In addition to reductions in HLA I and II (or MHC I and II), the hypoimmunogenic cells outlined herein have reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting hypoimmunogenic cells "evade" immune macrophages and natural pathways due to the expression of one or more CD47 transgenes.

[0429] J. Maintenance of hypoimmunogenic pluripotent stem cells Once low immunogenic pluripotent stem cells are generated, they can be maintained in an undifferentiated state, as is known with respect to the maintenance of iPSCs. For example, cells can be cultured on Matrigel using a culture medium that prevents differentiation while maintaining pluripotency. In addition, they can be kept in the culture medium under conditions that maintain pluripotency.

[0430] K. Differentiation of hypoimmunogenic pluripotent stem cells This technology provides low immunogenic pluripotent cells that can be differentiated into different cell types for subsequent transplantation into a target. As will be understood by those skilled in the art, the method for differentiation depends on the desired cell type using known techniques. The cells can be differentiated in suspension and then placed in a gel matrix form such as Matrigel, gelatin, or fibrin / thrombin form to facilitate cell viability. In some cases, differentiation is assayed, as is known in the art, by generally evaluating the presence of cell-specific markers.

[0431] In some embodiments, hypoimmunogenic pluripotent cells are differentiated into hepatocytes to address loss of hepatocyte function or cirrhosis. Several techniques exist that can be used to differentiate hypoimmunogenic pluripotent cells into hepatocytes. For example, Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb See et al., Hepatology 51:297-305 (2010), and Asgari et al., Stem Cell Rev:493-504 (2013), all of which are expressly incorporated herein by reference, in whole, particularly with respect to techniques and reagents for differentiation. Differentiation is assayed as is known in the art by assessing the presence of hepatocyte-related 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.

[0432] In some embodiments, low immunogenic pluripotent cells are differentiated into pancreatic beta-like cells or islet organoids for transplantation to address type 1 diabetes mellitus (TlDM). The cell system is a promising method for addressing TlDM. See, for example, Ellis et al., doi / 10.1038 / nrgastro.2017.93 (incorporated herein by reference). In addition, Pagliuca et al. report on the successful differentiation of β cells from human iPSCs (see doi / 10.106 / j.cell.2014.09.040 (in whole by reference, particularly with respect to the methods and reagents outlined in the same document for the large-scale production of functional human β cells from human pluripotent stem cells)). Furthermore, Vegas et al. demonstrate the production of human β cells from human pluripotent stem cells, followed by inclusion to avoid host immune rejection (doi :10.1038 / nm.4030 (The entire reference thereto is incorporated herein by reference, particularly with respect to the methods and reagents outlined therein for the large-scale production of functional human β-cells from human pluripotent stem cells).

[0433] Differentiation can be assayed, as is known in the art, by evaluating the presence of β-cell-related or specific markers, including but not limited to insulin. Differentiation can also be measured functionally, such as by measuring glucose metabolism. For general information, see Murarō et al., doi:10.1016 / j.cels.2016.09.002 (the entire work is incorporated herein by reference, particularly with respect to the biomarkers outlined therein).

[0434] In some embodiments, low immunogenic pluripotent stem cells are differentiated into retinal pigment epithelium (RPE) to address eye diseases that threaten vision. Human pluripotent stem cells were differentiated into RPE cells using the technique outlined in Kamao et al., Stem Cell Reports 2014:2:205-18 (the entire work is incorporated herein by reference, particularly with respect to the methods and reagents outlined in that work regarding the differentiation technique and reagents). See also Mandai et al., doi:10.1056 / NEJMoa1608368 (the entire work is also incorporated by reference, with respect to the technique for generating sheets of RPE cells and transplanting them into patients).

[0435] Differentiation can be assayed, as is known in the art, by generally evaluating the presence of RPE-related and / or specific markers, or by functional measurement. See, for example, Kamao et al., doi:10.1016 / j.stemcr.2013.12.007 (the entire work is incorporated herein by reference, particularly with respect to the markers outlined in the first paragraph of the Results section).

[0436] In some embodiments, low immunogenic pluripotent cells are differentiated into cardiomyocytes to address cardiovascular disease. Techniques for differentiating hiPSCs into cardiomyocytes are known in the art. Differentiation can be assayed, as is known in the art, by assessing the presence of cardiomyocyte-related or specific markers, or by functional measurement. See, for example, Loh et al., doi:10.1016 / j.cell.2016.06.001 (the entire work is incorporated herein by reference, particularly with respect to methods for differentiating stem cells, including cardiomyocytes).

[0437] In some embodiments, low immunogenic pluripotent cells are differentiated into endothelial colony-forming cells (ECFCs) to form new blood vessels to address peripheral artery disease. Techniques for differentiation into endothelial cells are known; see, for example, Prasain et al., doi:10.1038 / nbt.3048 (the entire work is referenced, in part, with respect to methods and reagents for generating endothelial cells from human pluripotent stem cells, and with respect to transplantation techniques). Differentiation can be assayed, as is known in the art, by assessing the presence of endothelial cell-related or specific markers, or by functional measurement.

[0438] In some embodiments, low immunogenic pluripotent cells are differentiated into thyroid progenitor cells and thyroid follicular organoids capable of secreting thyroid hormones to address autoimmune thyroiditis. Techniques for differentiation into thyroid cells are known in the art. See, for example, Kurmann et al., doi:10.106 / j.stem.2015.09.004 (the entire work is expressly incorporated herein by reference, particularly with respect to methods and reagents for generating thyroid cells from human pluripotent stem cells and transplantation techniques). Differentiation is generally evaluated for the presence of thyroid cell-related or specific markers. It can be assayed in the manner known in the art, either by means of or by functional measurement.

[0439] 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 Natl Acad Sci USA, 2019, 116(21), 10441-10446.

[0440] L. Administration of differentiated hypoimmunogenic cells 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 end use of these cells. Generally, the cells outlined can be transplanted in a patient either intravenously or by injection at a specific site. When transplanted at a specific site, the cells may be turbidified in a gel matrix to prevent dispersion during their establishment.

[0441] In some embodiments, methods for treating patients requiring cell therapy are provided herein, comprising administering a population of differentiated cells, including differentiated cells generated from stem cells conditionally expressing an exogenous immunosuppressive factor. In useful embodiments, methods for treating patients requiring cell therapy are provided herein, comprising administering a population of differentiated cells, including differentiated cells generated from stem cells conditionally expressing exogenous human CD47.

[0442] In some embodiments, a method for treating a patient requiring cell therapy involves administering a population of differentiated cells, including differentiated cells generated from stem cells that conditionally express low-immunity factors. In many embodiments, the differentiated cells are generated from stem cells that conditionally express essential factors.

[0443] 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 end use of these cells. Generally, the cells outlined herein can be transplanted in a patient either intravenously or by injection at a specific site. When transplanted at a specific site, the cells may be turbidified in a gel matrix to prevent dispersion during their establishment.

[0444] M. Exemplary Embodiments 1. Safety switches for controlling immunosuppressive factors In some embodiments, methods for controlling the immunogenicity of cells are provided herein, the methods comprising (a) obtaining isolated cells; (b) producing engineered cells by introducing into the isolated cells a nucleic acid comprising (i) an inducible RNA polymerase promoter operably linked to an shRNA sequence targeting an immunosuppressive factor, and (ii) a promoter (e.g., a constitutive promoter) operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter; and (c) exposing the engineered cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the cells. In some embodiments, the methods further comprise administering the engineered cells to a target prior to step (c).

[0445] In some embodiments, the method involves introducing a single construct into isolated cells comprising (i) an inducible RNA polymerase promoter operably ligated to an shRNA sequence targeting an immunosuppressive factor, and (ii) a promoter (e.g., a constitutive promoter) operably ligated to a transactivator element corresponding to the inducible RNA polymerase promoter. In some embodiments, the construct is ligated from the 5' end to the 3' end. It includes an inducible RNA polymerase promoter, an shRNA sequence targeting an immunosuppressive factor, a promoter (e.g., a constitutive promoter), and a transactivator element.

[0446] In some embodiments, the first construct comprises a nucleic acid comprising an inducible RNA polymerase promoter operably ligated to an shRNA sequence targeting an immunosuppressive factor, and the second construct comprises a nucleic acid comprising a promoter (e.g., a constitutive promoter) operably ligated to a transactivator element.

[0447] In some embodiments, isolated cells are engineered to exogenously express immunosuppressive factors. In some embodiments, isolated cells overexpress immunosuppressive factors in the absence of an exogenous factor that activates a transactivator element.

[0448] In some embodiments, the inducible RNA polymerase promoter of the construct is the U6Tet promoter.

[0449] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the immunosuppressive factor is CD47.

[0450] In some embodiments, the promoter described above is a constitutive promoter. In some embodiments, the constitutive promoter of the construct is selected from the group consisting of the eukaryotic elongation factor 1 α1 (EF1A) promoter, the eukaryotic elongation factor 1 α1 short-chain (EFS) promoter, the cytomegalovirus early enhancer / promoter (CMV promoter), the CMV early enhancer fused to modified chicken β-actin (CAGGS) promoter (also called the CAG promoter), the monkey virus 40 (SV40) promoter, the copia transposon (COPIA) promoter, the actin 5C (ACT5C) promoter, the tetracycline-responsive promoter element (TRE promoter), the CMV early enhancer fused to modified chicken β-actin (CBh) promoter, the phosphoglycerate kinase 1 (PGK) promoter, and the ubiquitin C (UBC) promoter.

[0451] In a particular embodiment, the construct comprises a U6Tet promoter, a CD47-targeting shRNA sequence, an EF1a promoter, and a Tet repressor element from its 5' to 3' end, wherein the exogenous factor is a tetracycline or a derivative thereof.

[0452] In some embodiments, one of the constructs outlined herein further includes a vector backbone for lentiviral expression.

[0453] In some embodiments, the isolated cells are isolated mammalian cells. In some embodiments, the isolated cells are isolated human cells.

[0454] In some embodiments, the isolated human cells further include deletion or reduced expression of MHC class I human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated human cells further include deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated human cells further include deletion or reduced expression of MHC class I and MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, the isolated human cells are CIITA This further includes deletion or reduction of expression. In some embodiments, isolated human cells further include deletion or reduction of expression of B2M. In some embodiments, isolated human cells further include deletion or reduction of expression of NLRC5. In some embodiments, isolated human cells are low immunogenic.

[0455] In some embodiments, isolated human cells are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, adult stem cells, and differentiated cells. In some embodiments, differentiated cells are selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0456] In another embodiment, a construct is provided herein comprising, from its 5' to 3' end, an inducible RNA polymerase promoter, an immunosuppressive factor-targeting shRNA sequence, a constitutive promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter. In some embodiments of the construct, the inducible RNA polymerase promoter is the U6Tet promoter.

[0457] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the immunosuppressive factor is CD47.

[0458] In some embodiments, the constitutive promoter of the construct is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0459] In some embodiments, the construct includes a U6Tet promoter, a CD47-targeting shRNA sequence, an EF1a promoter, and a Tet repressor element from the 5' end to the 3' end.

[0460] In some embodiments, the nucleic acid or construct also includes a vector backbone for lentiviral expression.

[0461] Compositions comprising isolated cells containing any one of the constructs described are also provided herein. In some embodiments of the composition, the isolated cells are exposed to an exogenous factor for activating the transactivator element. In some embodiments, the isolated cells described above are engineered to exogenously express an immunosuppressive factor. In certain embodiments, the isolated cells overexpress an immunosuppressive factor in the absence of an exogenous factor for activating the transactivator element.

[0462] In some embodiments, the isolated cells are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

[0463] Furthermore, compositions are also provided that include isolated differentiated cells prepared by culturing any of the stem cells described herein under differentiation conditions for the production of differentiated cells. In some embodiments, the differentiation conditions are suitable for differentiating stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0464] A method for treating a patient requiring cell therapy is provided herein, the method comprising (a) administering one of the compositions described to the patient, and (b) exposing the composition to an exogenous factor for activating an inducible RNA polymerase promoter, thereby controlling the cellular immunogenicity of the composition.

[0465] In some embodiments, methods for controlling the immunogenicity of cells are provided herein, the methods comprising (a) obtaining isolated cells; (b) introducing into the isolated cells a nucleic acid encoding an inducible degron element operably linked to an immunosuppressive factor, or a nucleic acid encoding an immunosuppressive factor operably linked to an immunosuppressive factor, to produce engineered cells; and (c) exposing the engineered cells to an exogenous factor for activating the inducible degron element, thereby controlling the immunogenicity of the cells.

[0466] In some embodiments, the method further includes administering the manipulated cells to the subject prior to step (c). In some embodiments, the inducible degron element is linked to an immunosuppressant by a flexible linker.

[0467] In some embodiments, the inducible degron element is located at the N-terminus of the immunosuppressant. In some embodiments, the inducible degron element is located at the C-terminus of the immunosuppressant. In some embodiments, the transcription of the nucleic acid described is controlled by a promoter, such as a constitutive promoter. In some embodiments, constructs comprising the nucleic acid outlined above are provided herein.

[0468] In some embodiments, the constitutive promoter in the construct is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0469] In some embodiments, the flexible linker is (GSG) n (Sequence ID 3), (GGGS) n (Sequence ID 1), and (GGGSGGGS) n Selected from the group consisting of (Sequence ID 2), where n is between 1 and 10.

[0470] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the immunosuppressive factor gene is CD47.

[0471] In some embodiments, the degron element is selected from the group consisting of ligand-inducible degron elements, peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements.

[0472] In some embodiments, the ligand-inducible degron element is selected from small molecule co-blocking (SMASH) degron elements, shield-1 responsive degron elements, auxin-responsive degron elements, and rapamycin-responsive degron elements. In some embodiments, the ligand-inducible degron element is a small molecule co-blocking (SMASH) degron element, and the exogenous factor is asunaprevir.

[0473] In some embodiments, the construct further includes 5' homology arms and 3' homology arms for targeted incorporation into the safe harbor locus. The Haber locus is selected from the group consisting of the AAVS1 locus, CLBYL locus, CXCR4 locus, Rosa26 locus, and CCR5 locus.

[0474] In some embodiments, the isolated cells are isolated mammalian cells. In some embodiments, the isolated cells are isolated human cells.

[0475] In some embodiments, isolated human cells further include deletion or reduced expression of MHC class I human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of both MHC class I and MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of CIITA. In some embodiments, isolated human cells further include deletion or reduced expression of B2M. In some embodiments, isolated human cells further include deletion or reduced expression of NLRC5. In some embodiments, isolated human cells are low immunogenic.

[0476] In some embodiments, isolated human cells are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, adult stem cells, and differentiated cells. In some embodiments, differentiated cells are selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0477] In one embodiment, a construct is provided herein comprising, from the 5' end to the 3' end, a promoter (e.g., a constitutive promoter), an inducible degron element, an optional sequence encoding a flexible linker, and an immunosuppressant gene. In another embodiment, a construct is provided herein comprising, from the 5' end to the 3' end, a promoter (e.g., a constitutive promoter), an immunosuppressant gene, an optional sequence encoding a flexible linker, and an inducible degron element.

[0478] In some embodiments, the constitutive promoter is selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

[0479] In some embodiments, the flexible linker is (GSG) n (Sequence ID 3), (GGGS) n (Sequence ID 1), and (GGGSGGGS) n Selected from the group consisting of (Sequence ID 2), where n is between 1 and 10.

[0480] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0481] In some embodiments, the degron element is selected from the group consisting of ligand-inducible degron elements, peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements. In some embodiments, the ligand-inducible degron element is selected from small molecule co-blocking (SMASH) degron elements, shield-1 responsive degron elements, auxin-responsive degron elements, and rapamycin-responsive degron elements. In a particular embodiment, the ligand-inducible degron element is a small molecule co-blocking (SMASH) degron element.

[0482] In some embodiments, the construct further includes 5' homology arms and 3' homology arms for target integration into intragenomic safe harbor loci. In some embodiments, the intragenomic safe harbor loci are selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5 loci.

[0483] Compositions comprising isolated cells containing any one of the constructs described are provided herein. In some embodiments, the isolated cells are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

[0484] Furthermore, compositions are also provided that include isolated differentiated cells prepared by culturing any of the stem cells described herein under differentiation conditions for the production of differentiated cells. In some embodiments, the differentiation conditions are suitable for differentiating stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

[0485] A method for treating a patient requiring cell therapy is provided herein, the method comprising (a) administering one of the compositions described to the patient, and (b) exposing the composition to an exogenous factor for activating an inducible degron element promoter, thereby controlling the cellular immunogenicity of the composition.

[0486] In one embodiment, a method for controlling the immunogenicity of cells is provided herein, the method comprising (a) obtaining isolated cells; (b) introducing into the isolated cells a first construct comprising (i) a first promoter (e.g., a constitutive promoter) and an immunosuppressant gene from the 5' to the 3' end; (ii) a second construct comprising a second promoter (e.g., a constitutive promoter) and a nucleic acid sequence encoding Cas9 or a variant thereof from the 5' to the 3' end; and (ii) a transactivator element corresponding to an inducible RNA polymerase promoter, an immunosuppressant-targeting guide RNA (gRNA) sequence, a third promoter (e.g., a constitutive promoter), and an inducible RNA polymerase promoter from the 5' to the 3' end; and (c) exposing the engineered cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the cells. In some embodiments, the method further comprises administering the engineered cells to a target prior to step (c).

[0487] In some embodiments, the inducible RNA polymerase promoter of the third construct is the U6Tet promoter, the transactivator element is the Tet repressor element (also referred to as the Tet-On transactivator), and the exogenous factor is tetracycline or a derivative thereof.

[0488] In some embodiments, the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the immunosuppressive factor is CD47.

[0489] In some embodiments, the first constitutive promoter, the second constitutive promoter, and the third constitutive promoter are the EF1A promoter, the EFS promoter, the CMV promoter, the CAGGS promoter, the SV40 promoter, the COPIA promoter, the ACT5C promoter, the TRE promoter, the CBh promoter, and the PGK promoter. Selected from the group consisting of , and the UBC promoter.

[0490] In some embodiments, the isolated cells are isolated mammalian cells. In some embodiments, the isolated cells are isolated human cells.

[0491] In some embodiments, isolated human cells further include deletion or reduced expression of MHC class I human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of both MHC class I and MHC class II human leukocyte antigens compared to unmodified human cells. In some embodiments, isolated human cells further include deletion or reduced expression of CIITA. In some embodiments, isolated human cells further include deletion or reduced expression of B2M. In some embodiments, isolated human cells further include deletion or reduced expression of NLRC5. In some embodiments, isolated human cells are low immunogenic.

[0492] In some embodiments, isolated human cells are selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

[0493] In another embodiment, a composition is provided herein comprising isolated cells containing a DNA-targeted nuclease system for controlling the immunogenicity of cells, the composition comprising: (a) a first element comprising a first promoter (e.g., a constitutive promoter) and an immunosuppressant gene from the 5' to the 3' end; (b) a second element comprising a second promoter (e.g., a constitutive promoter) and a nucleic acid sequence encoding Cas9 or a variant thereof from the 5' to the 3' end; and (c) a third element comprising an inducible RNA polymerase promoter, a guide RNA (gRNA...

Claims

1. A method for controlling the immunogenicity of manipulated cells, wherein the method is (a) Obtaining isolated cells, (b) To produce engineered cells by introducing into the isolated cells (i) a nucleic acid containing an inducible RNA polymerase promoter operably linked to an shRNA sequence targeting an immunosuppressive factor, and (ii) a nucleic acid containing a promoter operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter, (c) Exposing the manipulated cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the manipulated cells, The method, including the method described above.

2. A method for controlling the immunogenicity of manipulated cells, wherein the method is (a) Obtaining isolated cells, (b) Producing engineered cells by introducing nucleic acids into the isolated cells, which include (i) a sequence encoding an inducible degron element operably linked to an immunosuppressive factor, or (ii) a sequence encoding an immunosuppressive factor operably linked to an inducible degron element. (c) Exposing the manipulated cells to an exogenous factor for activating the inducible degron element, thereby controlling the immunogenicity of the manipulated cells, The method, including the method described above.

3. A method for controlling the immunogenicity of manipulated cells, (a) Obtaining isolated cells, (b) To the isolated cells, (i) A first construct comprising a first promoter and an immunosuppressive factor gene from the 5' end to the 3' end, (ii) A second construct comprising a nucleic acid sequence encoding a second promoter and Cas9 or a variant thereof at the 5' end to the 3' end, and (ii) A third construct comprising, from the 5' end to the 3' end, an inducible RNA polymerase promoter, a guide RNA (gRNA) sequence targeting the immunosuppressive factor, a third promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter, Introducing and (c) Exposing the manipulated cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the manipulated cells, The method, including the method described above.

4. A method for controlling the immunogenicity of manipulated cells, wherein the method is (a) Obtaining isolated cells, (b) To produce engineered cells by introducing into the isolated cells (i) a nucleic acid containing an inducible RNA polymerase promoter operably linked to an immune signaling factor gene, and (ii) a nucleic acid containing a promoter operably linked to a transactivator element corresponding to the inducible RNA polymerase promoter, (c) Exposing the manipulated cells to an exogenous factor for activating the transactivator element, thereby controlling the immunogenicity of the manipulated cells, The method, including the method described above.

5. The claim further includes administering the manipulated cells before step (c). The method described in any one of items 1 to 4.

6. The method according to claim 1 or 5, wherein step (b) involves introducing into the isolated cells a single nucleic acid construct comprising (i) the inducible RNA polymerase promoter operably linked to the shRNA sequence targeting the immunosuppressive factor, and (ii) the promoter operably linked to the transactivator element.

7. The method according to claim 6, wherein the construct comprises the inducible RNA polymerase promoter, the shRNA sequence, the promoter, and the transactivator element from the 5' end to the 3' end.

8. The method according to claim 4 or 5, wherein step (b) involves introducing into the isolated cells a single nucleic acid construct comprising (i) the inducible RNA polymerase promoter operably linked to the immune signaling factor gene, and (ii) the promoter operably linked to the transactivator element.

9. The method according to any one of claims 4, 5, or 8, wherein the construct comprises the inducible RNA polymerase promoter, the immune signaling factor gene, the promoter, and the transactivator element from the 5' end to the 3' end.

10. The method according to any one of claims 1 or 5 to 7, wherein the isolated cells are manipulated to exogenously express the immunosuppressive factor.

11. The method according to any one of claims 1, 5 to 7, or 10, wherein the isolated cells overexpress the immunosuppressive factor in the absence of the exogenous factor that activates the transactivator element.

12. The method according to any one of claims 1, 5 to 7, or 10 to 11, wherein the inducible RNA polymerase promoter is the U6Tet promoter.

13. The method according to claim 3 or 5, wherein the inducible RNA polymerase promoter is a U6Tet promoter, the transactivator element is a Tet repressor element, and the exogenous factor is tetracycline or a derivative thereof.

14. The method according to any one of claims 4, 5, or 8-9, wherein the inducible RNA polymerase promoter is a TRE promoter, the transactivator element is a Tet-On element, and the exogenous factor is tetracycline or a derivative thereof.

15. The method according to claim 2 or 5, wherein a flexible linker connects the inducible degron element to the immunosuppressive factor.

16. The aforementioned flexible linker, (GSG) ｎ (Sequence ID 3), (GGGS) ｎ (Sequence ID 1), and (GGGGGGGG) ｎ The method according to claim 15, wherein a selection is made from the group consisting of (Sequence ID 2), and in the sequence, n is 1 to 10.

17. The method according to any one of claims 1, 5, or 15-16, wherein step (b) involves introducing a single nucleic acid construct comprising a promoter operably linked to the nucleic acid into the isolated cells.

18. The method according to any one of claims 1 to 4 or 17, wherein the promoter is a constitutive promoter selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

19. The method according to any one of claims 3, 5, or 13, wherein the first promoter, the second promoter, and / or the third promoter are constitutive promoters independently selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

20. The method according to any one of claims 1 to 3 or 5 to 19, wherein the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

21. The method according to any one of claims 1, 5 to 14, or 17 to 20, wherein the construct comprises a U6Tet promoter, a CD47-targeting shRNA sequence, an EF1a promoter, and a Tet repressor element from its 5' end to its 3' end, and the exogenous factor is tetracycline or a derivative thereof.

22. The method according to any one of claims 1, 6 to 14, or 17 to 20, wherein the construct further comprises a vector backbone for lentiviral expression.

23. The method according to any one of claims 2, 5, or 8 to 20, wherein the inducible degron element is selected from the group consisting of ligand-inducible degron elements, peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements.

24. The method according to claim 23, wherein the ligand-inducible degron element is selected from a small molecule co-blocking (SMASH) degron element, a shield-1 responsive degron element, an auxin-responsive degron element, and a rapamycin-responsive degron element.

25. The method according to claim 23 or 24, wherein the ligand-inducible degron element is a small molecule co-blocking (SMASH) degron element, and the exogenous factor is asunaprevir.

26. The method according to any one of claims 17-18, 20, or 23-25, wherein the construct further comprises a 5' homology arm and a 3' homology arm for target incorporation to a safe harbor locus selected from the group consisting of the AAVS1 locus, the CLBYL locus, the CXCR4 locus, the Rosa26 locus, and the CCR5 locus.

27. The method according to any one of claims 1 to 4 and 5 to 26, wherein the isolated cells are isolated human cells further comprising deletion or reduced expression of MHC class I human leukocyte antigen and / or deletion or reduced expression of MHC class II human leukocyte antigen compared to unmodified human cells.

28. The method according to any one of claims 1 to 4 and 5 to 27, wherein the isolated human cells further comprise deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

29. The method according to any one of claims 1 to 4 and 5 to 28, wherein the isolated human cells are low immunogenic and are either stem cells or differentiated cells thereof, wherein the stem cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, and adult stem cells, and the differentiated cells are selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

30. The method according to claim 29, wherein the differentiated cells are pancreatic cells.

31. A construct comprising, from its 5' end to its 3' end, an inducible RNA polymerase promoter, an shRNA sequence targeting an immunosuppressive factor, a constitutive promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter.

32. A construct comprising, from the 5' end to the 3' end, an inducible RNA polymerase promoter, an immune signaling factor gene, a promoter, and a transactivator element corresponding to the inducible RNA polymerase promoter.

33. The construct according to claim 31, wherein the inducible RNA polymerase promoter is the U6Tet promoter.

34. The construct according to claim 32, wherein the inducible RNA polymerase promoter is a TRE promoter.

35. The construct according to claim 31 or 33, wherein the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

36. The construct according to claim 32 or 34, wherein the immune signaling factor is selected from the group consisting of B2M, MIC-A, MIC-B, HLA-A, HLA-B, HLA-C, RFXANK, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D.

37. The construct according to any one of claims 31 to 36, wherein the constitutive promoter is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

38. A construct according to any one of claims 31, 33, 35, or 37, comprising a U6Tet promoter, a CD47-targeting shRNA sequence, an EF1a promoter, and a Tet repressor element from the 5' end to the 3' end.

39. A construct according to any one of claims 32, 34, 36, or 37, comprising a TRE promoter, an immune signaling factor gene, an EF1a promoter, and a Tet-On element from the 5' end to the 3' end.

40. The construct according to any one of claims 31 to 39, further comprising a vector backbone for lentiviral expression.

41. A composition comprising isolated cells containing the construct according to any one of claims 31 to 40.

42. A composition comprising isolated cells comprising a construct according to any one of claims 31, 33, or 35-38, wherein the isolated cells are manipulated to exogenously express the immunosuppressive factor.

43. The composition according to claim 42, wherein the isolated cells overexpress the immunosuppressive factor in the absence of the exogenous factor that activates the transactivator element.

44. The composition according to any one of claims 40 to 43, wherein the isolated cells are exposed to an exogenous factor for activating the transactivator element.

45. The composition according to any one of claims 41 to 44, wherein the isolated cells are stem cells selected from the group consisting of embryonic stem cells, pluripotent stem cells, and adult stem cells.

46. A composition comprising isolated differentiated cells prepared by culturing the stem cells described in claim 45 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

47. A method for treating patients who require cell therapy, (a) Administering the composition described in claim 46 to a patient, (b) Exposing the composition to an exogenous factor for activating the inducible RNA polymerase promoter, thereby controlling the immunogenicity of the composition to the cells, The method, including the method described above.

48. Pluripotent stem cells comprising (i) reduction or silencing of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) a factor selected from the group consisting of inducible shRNA targeting CD47, inducible degron elements that regulate CD47, or SMASH degron elements that regulate CD47.

49. Pluripotent stem cells comprising (i) reduced or silencing expression of B2M and CIITA, (ii) overexpression of CD47, and (iii) a factor selected from the group consisting of an inducible shRNA targeting CD47, an inducible degron element that regulates CD47, or a SMASH degron element that regulates CD47.

50. Pluripotent stem cells comprising (i) reduced or silencing expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, (iii) Cas9 or a variant thereof, and (iv) inducible guide RNA targeting CD47.

51. Pluripotent stem cells comprising (i) reduced or silencing of B2M and CIITA expression, (ii) overexpression of CD47, (iii) Cas9 or a variant thereof, and (iv) inducible guide RNA targeting CD47.

52. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an inducible proteolytic system for regulating CD47 expression, selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolytic chimeric molecules, and antibodies for targeted degradation.

53. Pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) an inducible proteolytic system for regulating CD47 expression, selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolytic chimeric molecules, and antibodies for targeted degradation.

54. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an RNA regulatory system for regulating CD47 expression, selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

55. Pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) an RNA regulatory system for regulating CD47 expression, selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

56. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) a DNA regulatory system for regulating CD47 expression, selected from the group consisting of tissue-specific promoter expression systems, inducible promoter expression systems, molecularly controlled riboswitch systems, and inducible nuclease-based genome editing systems.

57. Pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) a DNA regulatory system for regulating CD47 expression, selected from the group consisting of tissue-specific promoter expression systems, inducible promoter expression systems, molecularly controlled riboswitch systems, and inducible nuclease-based genome editing systems.

58. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) overexpression of CD47, and (iii) an inducible system for regulating the expression of CD47.

59. Pluripotent stem cells comprising (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) an inducible system for regulating CD47 expression.

60. Differentiated cells derived from pluripotent stem cells according to any one of claims 48 to 59, wherein the differentiated cells are selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

61. From the 5' end to the 3' end, there is a promoter, an inducible degron element, and a flexible linker. A construct comprising an optional sequence encoding and an immunosuppressive factor gene.

62. A construct comprising a promoter, an immunosuppressive factor gene, an optional sequence encoding a flexible linker, and an inducible degron element from the 5' end to the 3' end.

63. The construct according to claim 61 or 62, wherein the promoter is a constitutive promoter selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

64. The aforementioned flexible linker, (GSG) ｎ (Sequence ID 3), (GGGS) ｎ (Sequence ID 1), and (GGGGGGGG) ｎ A construct according to any one of claims 61 to 63, selected from the group consisting of (Sequence ID 2), wherein n in the sequence is 1 to 10.

65. The construct according to any one of claims 61 to 64, wherein the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

66. The construct according to any one of claims 61 to 65, wherein the inducible degron element is selected from the group consisting of ligand-inducible degron elements, inducible peptidolytic degron elements, and peptidolytic proteolysis-inducible chimeric molecule (PROTAC) elements.

67. The construct according to claim 66, wherein the ligand-inducible degron element is selected from a small molecule co-blocking (SMASH) degron element, a shield-1 responsive degron element, an auxin-responsive degron element, and a rapamycin-responsive degron element.

68. The construct according to any one of claims 61 to 67, further comprising a 5' homology arm and a 3' homology arm for target integration to an intragenomic safe harbor locus selected from the group consisting of the AAVS1 locus, the CLBYL locus, the CXCR4 locus, the Rosa26 locus, and the CCR5 locus.

69. A composition comprising isolated cells containing the construct according to any one of claims 61 to 68.

70. The composition according to claim 69, wherein the isolated cells are stem cells selected from the group consisting of stem cells, embryonic stem cells, pluripotent stem cells, and adult stem cells.

71. A composition comprising isolated differentiated cells prepared by culturing the stem cells described in claim 70 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

72. A method for treating patients who require cell therapy, (a) Administering the composition according to claim 71 to the patient, (b) Exposing the composition to an exogenous factor for activating the inducible degron element, thereby controlling the immunogenicity of the composition to the cells, The method, including the method described above.

73. A composition comprising isolated cells containing a DNA-targeted nuclease system for controlling the immunogenicity of the cells, (a) A first element comprising a first promoter and an immunosuppressive factor gene from the 5' end to the 3' end, (b) A second element comprising a nucleic acid sequence encoding a second promoter and Cas9 or a variant thereof, from the 5' end to the 3' end, (c) A third element comprising an inducible RNA polymerase promoter, a guide RNA (gRNA) sequence targeting the immunosuppressive factor, a third promoter, and a transactivator element corresponding to the inducible promoter, from the 5' end to the 3' end, The composition comprising the above.

74. The composition according to claim 73, wherein the immunogenicity of the cells is controllable when the cells are exposed to an exogenous factor for inducing the activity of the transactivator element.

75. The composition according to claim 73 or 74, wherein the inducible RNA polymerase promoter is a U6Tet promoter, the transactivator element is a Tet repressor element, and the exogenous factor is tetracycline or a derivative thereof.

76. The composition according to any one of claims 73 to 75, wherein the immunosuppressive factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

77. The composition according to any one of claims 73 to 76, wherein the first promoter, the second promoter, and / or the third promoter are constitutive promoters independently selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

78. The composition according to any one of claims 73 to 77, wherein the isolated cells are isolated and manipulated human cells, further comprising deletion or reduction of expression of MHC class I human leukocyte antigen and / or deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

79. The composition according to claim 78, wherein the isolated human cells further comprise deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

80. The composition according to claim 78 or 79, wherein the isolated human cells are low immunogenic and are stem cells.

81. A composition comprising isolated differentiated cells prepared by culturing the stem cells described in claim 80 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

82. A method for treating patients who require cell therapy, (a) administering the composition described in claim 81, (b) Exposing the composition to an exogenous factor for activating the inducible RNA polymerase promoter, thereby controlling the immunogenicity of the composition to the cells, The method, including the method described above.

83. A composition comprising isolated mammalian cells, comprising modifications including recombinant nucleic acid sequences encoding a system for the conditional expression of one or more immunosuppressive factors.

84. A composition comprising isolated mammalian cells containing recombinant nucleic acid sequences encoding a system for the conditional expression of one or more immune signaling factors.

85. The composition according to claim 83, wherein the expression of the one or more immunosuppressive factors is controllable by exogenous factors.

86. The composition according to claim 84, wherein the expression of the one or more immune signaling factors is controllable by an exogenous factor.

87. The composition according to claim 83 or 85, wherein the system comprises an inducible proteolytic system for reducing the protein level of one or more immunosuppressive factors.

88. The composition according to claim 87, wherein the inducible proteolysis system is selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolysis-inducible chimeric molecule, and antibody for targeted degradation.

89. The composition according to claim 83 or 85, wherein the system comprises an RNA control system for controllably reducing the RNA level of one or more immunosuppressive factors.

90. The composition according to claim 89, wherein the RNA regulatory system is selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and an RNA-targeting nuclease system.

91. The composition according to claim 90, wherein the RNA regulatory system is controllable by a ligand-inducible transcription factor, a SynNotch receptor, or a ligand-controlled riboswitch.

92. The composition according to claim 83 or 85, wherein the system comprises a DNA control system for reducing the expression level of one or more immunosuppressive factors, selected from the group consisting of a tissue-specific promoter expression system, an inducible promoter expression system, a molecularly controlled riboswitch system, and an inducible nuclease-based genome editing system.

93. The composition according to claim 92, wherein the inducible promoter expression system comprises a U6Tet promoter and a Tet repressor element.

94. The composition according to claim 84 or 86, wherein the system comprises an inducible protein stabilization system for increasing the protein level of one or more immune signaling factors.

95. The composition according to claim 94, wherein the inducible protein stabilization system comprises a ligand-induced protein stabilization system and a low molecular weight inducible protein stabilization system.

96. The composition according to claim 84 or 86, wherein the system comprises an RNA regulatory system for increasing the RNA level of one or more immune signaling factors.

97. The composition according to claim 96, wherein the RNA regulatory system comprises a CRISPR activation (CRISPRRa) system.

98. The composition according to claim 84 or 86, wherein the system comprises a DNA control system for increasing the expression level of one or more immune signaling factors.

99. The composition according to claim 98, wherein the DNA control system comprises one or more DNA control systems selected from the group consisting of a CRISPR activation (CRISPRRa) system, a tissue-specific promoter, an inducible promoter, and a molecularly controlled riboswitch system.

100. The composition according to claim 92 or 99, wherein the tissue-specific promoter is selected from the group consisting of cardiac cell-specific promoters, hepatocyte-specific promoters, kidney cell-specific promoters, pancreatic cell-specific promoters, nerve cell-specific promoters, immune cell-specific promoters, mesenchymal cell-specific promoters, and endothelial cell-specific promoters.

101. The composition according to claim 99, wherein the inductive promoter comprises a TetOn system.

102. The composition according to claim 92 or 99, wherein the molecularly controlled riboswitch system comprises a theophylline-controlled riboswitch or a guanine-controlled riboswitch.

103. The composition according to claim 92, wherein the inducible nuclease-based genome editing system comprises one selected from the group consisting of CRISPR genome editing, inducible TALEN genome editing, inducible ZFN genome editing, and low-molecular-weight enhanced CRISPR-based genome editing, each comprising an inducible guide RNA targeting one or more immunosuppressive factors.

104. The composition according to any one of claims 83, 85, 87-93, 100, or 102-103, wherein the one or more immunosuppressive factors are selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

105. The composition according to any one of claims 84, 86, or 94-102, wherein the one or more immune signaling factors are selected from the group consisting of beta-2-microglobulin (B2M), MHC class I-related protein A (MIC-A), MHC class I-related protein B (MIC-B), HLA-A, HLA-B, HLA-C, RFXANK, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D.

106. The composition according to any one of claims 83 to 105, wherein the isolated mammalian cells are isolated and manipulated human cells further comprising deletion or reduction of expression of MHC class I human leukocyte antigen and / or deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

107. The composition according to claim 106, wherein the isolated and manipulated human cells further comprise deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

108. The composition according to any one of claims 106 to 107, wherein the isolated and manipulated human cells are low immunogenic and are stem cells.

109. A composition comprising isolated differentiated cells prepared by culturing the stem cells described in claim 108 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

110. A method for treating patients who require cell therapy, (a) administering the composition described in claim 109, (b) Exposing the composition to an exogenous factor for controlling the expression of one or more immunosuppressive factors, thereby controlling the immunogenicity of the composition to the cells, The method, including the method described above.

111. A construct comprising, at its 5' to 3' end, (1) a safety switch introduction gene, (2) a sequence encoding a ribosome skipping sequence and / or a linker, and (3) a low-immunity gene.

112. A construct comprising (1) a low-immunity gene, (2) a ribosome skipping sequence or linker, and (3) a safety switch introduction gene at the 5' to 3' end.

113. The construct according to claim 111 or 112, wherein the safety switch gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspace9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD16 gene, and CD30 gene.

114. The construct according to any one of claims 101 to 103, wherein the ribosome skipping sequence includes a sequence encoding an IRES sequence or a sequence encoding a 2A coding sequence.

115. The structure according to any one of claims 111 to 114, wherein the linker is selected from any one of the linkers provided in Table 3.

116. The construct according to any one of claims 111 to 115, wherein the hypoimmune gene is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

117. The construct according to any one of claims 111 or 113 to 116, further comprising a transcriptional regulatory element operably linked to the safety switch gene and a polyadenylated sequence at the 3' end of the hypoimmune gene, or a transcriptional regulatory element operably linked to the hypoimmune gene and a polyadenylated sequence at the 3' end of the safety switch gene.

118. Any of claims 111 to 117, wherein the transcriptional control element is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter. A structure as described in item 1.

119. The construct according to any one of claims 111 to 118, further comprising a vector backbone for lentiviral expression.

120. A method for delivering a construct into isolated cells, comprising transducing isolated cells with a lentiviral construct comprising the construct according to claim 119, and selecting engineered cells possessing the safety switch gene and the low-immunity gene.

121. Isolated cells or populations thereof comprising the construct according to any one of claims 111 to 119.

122. The isolated cells or population thereof according to claim 121, wherein the construct is introduced into a target gene locus.

123. The isolated cells or population thereof according to claim 121 or 122, wherein the target gene locus is a safe harbor locus selected from the group consisting of AAVS1, CLBYL, CXCR4, Rosa26, and CCR5, or an immune signaling locus selected from the group consisting of other ligands such as B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and NKG2D.

124. The isolated cells or population thereof according to any one of claims 121 to 123, wherein the isolated cells are isolated and engineered human cells further comprising deletion or reduced expression of MHC class I human leukocyte antigen and / or deletion or reduced expression of MHC class II human leukocyte antigen compared to unmodified human cells.

125. The isolated cells or population thereof according to any one of claims 121 to 124, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

126. The isolated cells or population thereof according to any one of claims 121 to 125, wherein the isolated cells are low immunogenic and are stem cells.

127. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 126 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

128. A method for treating a patient requiring cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 127.

129. A method for treating a patient, comprising activating a safety switch in a patient who has been previously administered differentiated cells or a population thereof according to claim 127.

130. At the 5' to 3' end, (1) a first homology arm homologous to the first endogenous sequence of the safe harbor locus, (2) a safety switch introduction gene, (3) a sequence encoding a ribosome skipping sequence and / or a linker, (4) a low-immunity gene, and (5) a polyadenylated sequence. , and (6) a construct for homology-directed repair into the safe harbor locus, comprising a second homology arm homologous to a second endogenous sequence of the safe harbor locus.

131. A construct for homology-directed repair into a safe harbor locus, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of an immune signaling locus; (2) a safety switch introduction gene; (3) a sequence encoding a ribosome skipping sequence and / or a linker; (4) a hypoimmune gene; (5) a polyadenylation sequence; and (6) a second homology arm homologous to a second endogenous sequence of the immune signaling locus.

132. At the 5' to 3' end, (1) a first homology arm homologous to the first endogenous sequence of the safe harbor locus, (2) a safety switch transgene, (3) a sequence encoding a ribosome skipping sequence or linker, and (4) an essential sequence. A construct for homology-directed repair into a safe harbor locus, comprising (5) a cellular factor gene, a polyadenylated sequence, and (6) a second homology arm homologous to a second endogenous sequence of the safe harbor locus.

133. At the 5' to 3' end, (1) a first homology arm homologous to the first endogenous sequence of the immune signaling locus, (2) a safety switch transgene, (3) a sequence encoding a ribosome skipping sequence or linker, and (4) an essential sequence. A construct for homology-directed repair into immune signaling, comprising (5) a cellular factor gene, and (6) a polyadenylated sequence, and a second homology arm homologous to a second endogenous sequence of the immune signaling locus.

134. A construct for homology-directed repair into an essential cell factor locus, comprising at its 5' to 3' end: (1) a first homology arm homologous to the first endogenous sequence of the essential cell factor locus; (2) a sequence encoding a linker; (3) a safety switch introduction gene; and (4) a second homology arm homologous to the second endogenous sequence of the essential cell factor locus.

135. The construct according to claim 130 or 131, wherein the hypoimmune gene is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

136. The construct according to claim 132 or 133, wherein the essential cellular factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

137. The construct according to claim 134, wherein the essential cellular factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

138. The construct according to any one of claims 130, 132, or 135-136, wherein the safe harbor locus is selected from the group consisting of the AAVS1 locus, the CLBYL locus, the CXCR4 locus, the Rosa26 locus, and the CCR5 locus.

139. The aforementioned immune signaling gene loci are B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 A construct according to any one of claims 131, 133, or 135-136, selected from the group consisting of / ULBP1, RAE11L / ULBP6, RAET1N / ULBP3, and other ligands of NKG2D.

140. The construct according to any one of claims 133, 136, or 139, wherein the immune signaling gene locus is selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, and HLA-E.

141. The construct according to any one of claims 130-133, 135-136, or 138-140, wherein the ribosome skipping sequence includes a sequence encoding an IRES sequence or a sequence encoding a 2A coding sequence.

142. The structure according to claim 141, wherein the 2A code sequence is selected from the group consisting of T2A, P2A, E2A, and F2A.

143. The construct according to any one of claims 130-133, 135-136, or 138-142, wherein the construct enables a targeted nuclease to cleave the safe harbor locus or the immune signaling locus, thereby allowing the construct to be recombined into the locus by homology-directed repair.

144. The construct according to claim 134 or 137, wherein the construct enables a targeted nuclease to cleave the essential cell factor locus, thereby allowing the construct to be recombined into the locus by homology-directed repair.

145. The construct according to any one of claims 130 to 144, further comprising a transcriptional regulatory element selected from the group consisting of the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter, located at the 5' end of the safety switch introduced gene.

146. The construct according to any one of claims 130 to 145, wherein the safety switch introduced gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspace9 gene, HER1 introduced gene, RQR8 introduced gene, CD20 introduced gene, CCR4 introduced gene, HER2 introduced gene, CD19 introduced gene, MUC1 introduced gene, EGFR introduced gene, GD2 introduced gene, PSMA introduced gene, CD16 introduced gene, and CD30 introduced gene.

147. The structure according to any one of claims 130 to 146, wherein the linker is selected from any one of the linkers provided in Table 3.

148. Isolated cells or population thereof comprising a safety switch gene and a hypoimmune gene incorporated into a safe harbor locus or an immune signaling locus, wherein the construct according to any one of claims 130, 135, 138, 141-143, or 145-147 is recombined into the endogenous safe harbor locus of the cell, or the construct according to any one of claims 131, 135, or 135-147 is recombined into the endogenous immune signaling locus of the cell.

149. A safety switch integrated into the safe harbor locus or immune signaling locus Isolated cells or population thereof, comprising a transgene and an essential cell factor gene, wherein the construct according to any one of claims 132, 136, 138, 141, 143, or 145-147 is recombined within the endogenous safe harbor locus of the cell, or the construct according to any one of claims 133, 136, 139-143, or 145-147 is recombined within the endogenous immune signaling locus of the cell, and the cell or population thereof is unable to express the essential cell factor from the endogenous locus.

150. The isolated cells or population thereof according to claim 148 or 149, wherein the isolated cells are isolated and engineered human cells further comprising deletion or reduction of expression of MHC class I human leukocyte antigen and / or deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

151. The isolated cells or population thereof according to any one of claims 148 to 150, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

152. The isolated cells or population thereof according to any one of claims 148 to 151, wherein the isolated cells are low immunogenic and are stem cells.

153. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 152 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

154. A method for treating a patient in need of cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 153.

155. A method for treating a patient, comprising activating a safety switch in a patient who has been previously administered differentiated cells or a population thereof as described in claim 153.

156. Homology-independent donor construct comprising, at the 5' to 3' end, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) a safety switch transgene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) a hypoimmune gene, (6) a polyadenylated sequence, and (7) a 3' LTR containing a right-side element (RE).

157. Homology-independent donor constructs comprising, at the 5' to 3' ends, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) a safety switch transgene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) an essential cell factor gene, (6) a polyadenylated sequence, and (7) a 3' LTR containing a right-side element (RE).

158. Homology-independent donor constructs comprising, at the 5' to 3' ends, (1) a 5' long-chain terminal repeat sequence (LTR) containing a left-side element (LE), (2) a splice acceptor-virus 2A peptide (SA-2A) element, (3) an essential cell factor gene, (4) a sequence encoding a ribosome skipping sequence or linker, (5) a safety switch introduction gene, (6) a polyadenylated sequence, and (7) a 3' LTR containing a right-side element (RE).

159. The aforementioned low-immunity genes are CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, I The structure according to claim 156, selected from the group consisting of L-35, FASL, Serpinb9, CCl21, and Mfge8.

160. The construct according to claim 157 or 158, wherein the essential cellular factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

161. The construct according to any one of claims 156 to 160, wherein the construct is configured to be incorporated into a target gene locus of an isolated cell to interfere with the expression of the target gene.

162. The construct according to any one of claims 156 to 161, wherein the safety switch introduced gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspace9 gene, HER1 introduced gene, RQR8 introduced gene, CD20 introduced gene, CCR4 introduced gene, HER2 introduced gene, CD19 introduced gene, MUC1 introduced gene, EGFR introduced gene, GD2 introduced gene, PSMA introduced gene, CD16 introduced gene, and CD30 introduced gene.

163. The construct according to any one of claims 156 to 162, wherein the target gene locus is an immune signaling gene locus selected from the group consisting of other ligands of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and NKG2D.

164. The construct according to any one of claims 156 to 162, wherein the target gene locus is an immune signaling gene locus selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, and HLA-E.

165. The construct according to any one of claims 156 to 164, wherein the target gene locus is a safe harbor locus selected from the group consisting of the AAVS1 locus, the CLBYL locus, the CXCR4 locus, the Rosa26 locus, and the CCR5 locus.

166. Isolated cells or a population of cells comprising the construct according to any one of claims 156 to 165, wherein the construct is incorporated into an endogenous target gene so as to interfere with the expression of the target gene in the isolated cells.

167. The isolated cells or population according to claim 166, wherein the isolated cells are unable to express the essential cell factor from the endogenous gene locus.

168. The isolated cells or population thereof according to claim 167, wherein the construct is incorporated into the target gene at a nuclease or transposase target site.

169. The isolated cells or population thereof according to any one of claims 166 to 168, wherein one allele of the target gene is destroyed by targeting with a nuclease or transposase.

170. Both alleles of the target gene are by the nuclease or the transposase An isolated cell or population thereof according to any one of claims 166 to 169, which is destroyed by targeted targeting.

171. The isolated cells or population thereof according to any one of claims 166 to 170, wherein the isolated cells are isolated and engineered human cells further comprising deletion or reduction of expression of MHC class I human leukocyte antigen and / or deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

172. The isolated cells or population thereof according to claim 171, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

173. The isolated cells or population thereof according to any one of claims 166 to 172, wherein the isolated cells are low immunogenic and are stem cells.

174. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 173 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

175. A method for treating a patient requiring cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 174.

176. A method for treating a patient, comprising activating the safety switch in the patient who has been previously administered differentiated cells or a population thereof according to claim 174 or 175.

177. Isolated cells or populations containing essential cell factor genes operably linked to sequences encoding linkers operably linked to safety switch-introduced genes.

178. The isolated cells or population thereof according to claim 177, wherein the essential cellular factors are selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

179. The isolated cells or population thereof according to claim 177 or 178, wherein the linker is selected from any one of the linkers provided in Table 3.

180. Isolated cells or populations thereof according to any one of claims 177 to 179, wherein the safety switch gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspace9 gene, HER1 gene, RQR8 gene, CD20 gene, CCR4 gene, HER2 gene, CD19 gene, MUC1 gene, EGFR gene, GD2 gene, PSMA gene, CD30 gene, and CD16 gene.

181. (1) A low-immune factor selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms, and (2) CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, G A recombinant peptide epitope fusion protein comprising a peptide epitope exposed on a surface of a different type from the low-immunity factor, selected from the group consisting of D2 epitopes, PSMA epitopes, CD16 epitopes, and CD30 epitopes.

182. A construct encoding a recombinant peptide epitope fusion protein, comprising: (1) a sequence encoding a low-immunity factor selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms; and (2) a sequence encoding a surface-exposed peptide epitope of a different type from the low-immunity factor, selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

183. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The protein according to claim 181 or the construct according to claim 182, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

184. The protein according to claim 181 or 183, wherein the low-immunity factor and / or the peptide epitope is located at the N-terminus of the fusion protein.

185. The protein according to any one of claims 181 or 183, further comprising a linker located at the N-terminus or C-terminus of the fusion protein, which links the low-immunity factor and the peptide epitope, wherein the linker is selected from any one of the linkers provided in Table 3.

186. The construct according to any one of claims 182 or 183, wherein the sequence encoding the low-immunity factor is located at 5' of the sequence encoding the peptide epitope, and / or the sequence encoding the peptide epitope is located at 5' of the sequence encoding the low-immunity factor.

187. The claim according to any one of claims 182 or 183, further comprising a sequence that links the sequence encoding the low-immunity factor and the sequence encoding the peptide epitope, and / or a sequence that encodes a linker located at the N-terminus or C-terminus of the fusion protein. Structure.

188. The structure according to claim 187, wherein the linker is selected from any one of the linkers provided in Table 3.

189. The construct according to any one of claims 182 to 184 or 186 to 188, further comprising a transcriptional regulatory element selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

190. A construct according to any one of claims 182-184 or 186-189, further comprising a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair.

191. A construct according to any one of claims 182-184 or 186-190, further comprising a vector backbone for lentiviral expression.

192. A method comprising transducing isolated cells with the construct described in claim 191, and selecting the isolated cells that express the recombinant peptide epitope fusion protein.

193. Isolated cells or populations thereof comprising a construct according to any one of claims 182-184 or 186-191.

194. The isolated cells or population thereof according to claim 193, wherein the isolated cells further comprise the absence or reduction of expression of MHC class I human leukocyte antigen and / or the absence or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

195. The isolated cells or population thereof according to any one of claims 193 to 194, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

196. The isolated cells or population thereof according to any one of claims 193 to 195, wherein the isolated cells are low immunogenic and are stem cells.

197. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 196 under differentiation conditions suitable for differentiation of stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

198. A method for treating a patient requiring cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 197.

199. A method for treating a patient, comprising administering an antibody that binds to the peptide epitope to a patient who has been previously administered differentiated cells or a population thereof as described in claim 197.

200. The method according to claim 199, wherein the antibody mediates ADCC or CDC.

201. A recombinant CD47 internal peptide epitope fusion protein comprising, from the N-terminus to the C-terminus, (1) a human CD47 fragment containing the IgV domain of CD47, (2) a first linker, (3) a heterologous peptide epitope, (4) a second linker, and (5) a human CD47 transmembrane domain.

202. The protein according to claim 201, wherein the human CD47 fragment containing the IgV domain comprises amino acid residues 1 to 127 of the human CD47 protein.

203. The protein according to claim 201 or 202, wherein the human CD47 transmembrane domain comprises amino acid residues 128 to 348 of the human CD47 protein.

204. The protein according to any one of claims 201 to 203, wherein the first linker and the second linker are selected from any one of the linkers provided in Table 3.

205. The protein according to any one of claims 201 to 204, wherein the peptide epitope is selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

206. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The protein according to claim 205, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

207. A construct comprising, at its 5' to 3' end, (1) a transcriptional regulatory element, (2) a sequence encoding a human CD47 fragment including the IgV domain of CD47, (3) a first linker, (4) a sequence encoding a peptide epitope, (5) a second linker, and (6) a sequence encoding a human CD47 fragment including a transmembrane domain and a C-terminus.

208. The construct according to 207, wherein the human CD47 fragment containing the IgV domain encodes amino acid residues 1 to 127 of the human CD47 protein.

209. The construct according to claim 207 or 208, wherein the human CD47 fragment encoding the transmembrane domain and the C-terminus comprises amino acid residues 128 to 348 of the human CD47 protein.

210. The structure according to any one of claims 207 to 209, wherein the first linker and the second linker are selected from any one of the linkers provided in Table 3.

211. The construct according to any one of claims 207 to 210, wherein the peptide epitope encoded by the sequence of (4) of the construct is selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

212. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The construct according to claim 211, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

213. The construct according to any one of claims 207 to 212, wherein the transcriptional control element is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

214. The construct according to any one of claims 207 to 213, further comprising a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair.

215. The construct according to any one of claims 207 to 214, further comprising a vector backbone for lentiviral expression.

216. A method comprising transducing isolated cells with the construct described in claim 215, and selecting the isolated cells that express the CD47 internal peptide epitope fusion protein.

217. Isolated cells or populations thereof comprising the construct according to any one of claims 207 to 215.

218. The isolated cells or population thereof according to claim 217, wherein the isolated cells are isolated and engineered human cells further comprising deletion or reduced expression of MHC class I human leukocyte antigen and / or deletion or reduced expression of MHC class II human leukocyte antigen compared to unmodified human cells.

219. The isolated cells or population thereof according to any one of claims 217 to 218, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

220. The isolated cells or population thereof according to any one of claims 217 to 219, wherein the isolated cells are low immunogenic and are stem cells.

221. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 220 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

222. A method for treating a patient in need of cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 221.

223. A method for treating a patient who has been previously administered differentiated cells or a population thereof according to claim 221, the method comprising administering to the patient an antibody that binds to the peptide epitope.

224. The method according to claim 223, wherein the antibody mediates ADCC or CDC.

225. A construct comprising (1) a transcriptional regulatory element, (2) an essential cell factor gene, (3) a post-transcriptional or post-translational regulatory element, and (4) a polyadenylated sequence.

226. The construct according to claim 225, wherein the essential cellular factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, and spliceosome subunit proteins.

227. The construct according to claim 225 or 226, wherein the transcriptional control element is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

228. The construct according to any one of claims 225 to 227, wherein the post-transcriptional regulatory element is an RNA regulatory system selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

229. The construct according to any one of claims 225 to 227, wherein the post-translational control element is an inducible proteolysis system selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolysis-inducible chimeric molecule, and antibody for targeted degradation.

230. Isolated cells containing recombinant essential cell factors under the control of post-transcriptional or post-translational regulatory elements, wherein the endogenous essential cell factor genes are inactivated and the expression of the recombinant essential cell factors is regulated by exogenous factors.

231. The isolated cell according to claim 230, wherein the essential cell factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit protein, proteasome subunit protein, and spliceosome subunit protein.

232. The isolated cells according to claims 230 to 231, wherein the post-transcriptional regulatory element is an RNA regulatory system selected from the group consisting of inducible shRNA, inducible siRNA, CRISPR interference (CRISPRi), and RNA-targeted nuclease systems.

233. Isolated cells according to any one of claims 230 to 232, wherein the post-translational regulatory element is an inducible proteolysis system selected from the group consisting of a small molecule co-blocking (SMASH) system, Shield-1 inducible degron, auxin inducible degron, IMid inducible degron, peptidolytic degron, proteolysis-inducible chimeric molecules, and antibodies for targeted degradation.

234. The isolated cells or population thereof according to any one of claims 230 to 233, wherein the isolated cells are autologous human cells or allogeneic human cells of the same species.

235. The isolated cells or population thereof according to claim 234, wherein the isolated cells are isolated and engineered human cells further comprising deletion or reduction of expression of MHC class I human leukocyte antigen and / or deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

236. The isolated cells or population thereof according to claim 234 or 235, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

237. The isolated cells or population thereof according to any one of claims 234 to 236, wherein the isolated cells are low immunogenic and selected from the group consisting of stem cells and differentiated cells.

238. A bisistronic construct comprising, at its 5' to 3' end, (1) a transcriptional regulatory element, (2) a sequence encoding a surface-exposed peptide epitope, (3) a ribosome skipping sequence, and (4) a sequence encoding a low-immunity factor.

239. The construct according to claim 238, wherein the low-immunity factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms.

240. The construct according to claim 238 or 239, wherein the peptide epitope exposed on the surface encoded by the sequence of (2) of the construct is selected from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope.

241. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The construct according to claim 240, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

242. The construct according to any one of claims 238 to 241, wherein the ribosome skipping sequence includes a sequence encoding an IRES sequence or a sequence encoding a 2A coding sequence.

243. The construct according to any one of claims 238 to 242, wherein the transcriptional control element is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

244. The construct according to any one of claims 238 to 243, further comprising a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-oriented repair.

245. The construct according to any one of claims 238 to 244, further comprising a vector backbone for lentiviral expression.

246. A method comprising transducing isolated cells with the construct described in claim 245, and selecting the isolated cells that express the low immunofactor and the peptide epitope.

247. Isolated cells or a population thereof comprising the construct according to any one of claims 238 to 245.

248. The isolated cells or population thereof according to claim 247, wherein the isolated cells further comprise the deletion or reduction of expression of MHC class I human leukocyte antigen and / or the deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

249. The isolated cells further comprise the deletion or reduction of expression of CIITA, B2M, and / or NLRC5, as described in any one of claims 247 to 248. That group.

250. The isolated cells or population thereof according to any one of claims 247 to 249, wherein the isolated cells are low immunogenic and are stem cells.

251. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 250 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

252. A method for treating a patient in need of cell therapy, comprising administering to the patient a differentiated cell or population thereof according to claim 251.

253. A method for treating a patient, comprising administering an antibody that binds to the peptide epitope to a patient who has been previously administered differentiated cells or a population thereof as described in claim 251.

254. The method according to claim 253, wherein the antibody mediates ADCC or CDC.

255. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) a safety switch gene, and (iii) a low-immunity factor gene, wherein the expression of the safety switch gene modulates the expression of the low-immunity factor gene.

256. Pluripotent stem cells comprising (i) reduced or silencing of B2M and CIITA expression, (ii) overexpression of CD47, (iii) a safety switch gene, and (iv) a low-immunity factor gene, wherein the expression of the safety switch gene regulates the expression of the low-immunity factor gene.

257. Pluripotent stem cells comprising (i) reduction or silencing of the expression of MHC class I molecules and / or MHC class II molecules, (ii) a safety switch, and (iv) a low-immunity factor, wherein the expression of the safety switch modulates the expression of the low-immunity factor.

258. Pluripotent stem cells comprising (i) reduced or silencing expression of B2M and CIITA, (ii) overexpression of CD47, (iii) a safety switch, and (iv) a low-immunity factor, wherein the expression of the safety switch modulates the expression of the low-immunity factor.

259. (i) reduction or silencing of MHC class I molecules and / or MHC class II molecules, and (ii) a low-immunity factor linked to a surface-exposed peptide epitope, wherein the peptide epitope is a CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope Pluripotent stem cells, selected from the group consisting of CD16 epitope and CD30 epitope, wherein the low-immunity factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms.

260. (i) reduction or silencing of B2M and CIITA expression, (ii) overexpression of CD47, and (iii) low immunofactors linked to surface-exposed peptide epitopes, wherein the peptide epitopes are CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD Pluripotent stem cells selected from the group consisting of two epitopes, PSMA epitopes, CD16 epitopes, and CD30 epitopes, wherein the low-immunity factor is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, Mfge8, and their membrane-bound forms.

261. A construct comprising, at its 5' to 3' end, (1) a safety switch introduction gene, (2) a sequence encoding a ribosome skipping sequence and / or a linker, and (3) an essential cell factor gene.

262. A construct comprising (1) an essential cell factor gene, (2) a ribosome skipping sequence or linker, and (3) a safety switch introduction gene at its 5' to 3' end.

263. The construct according to claim 261 or 262, wherein the safety switch introduced gene is selected from the group consisting of the HSVtk gene, cytosine deaminase gene, nitroreductase gene, purine nucleoside phosphorylase gene, horseradish peroxidase gene, iCaspace9 gene, HER1 introduced gene, RQR8 introduced gene, CD20 introduced gene, CCR4 introduced gene, HER2 introduced gene, CD19 introduced gene, MUC1 introduced gene, EGFR introduced gene, GD2 introduced gene, PSMA introduced gene, CD16 introduced gene, and CD30 introduced gene.

264. The construct according to any one of claims 261 to 263, wherein the ribosome skipping sequence includes a sequence encoding an IRES sequence or a sequence encoding a 2A coding sequence.

265. The structure according to any one of claims 261 to 264, wherein the linker is selected from any one of the linkers provided in Table 3.

266. The construct according to any one of claims 261 to 265, wherein the hypoimmune gene is selected from the group consisting of CD47, CD24, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

267. The construct according to any one of claims 261 or 263 to 266, further comprising a transcriptional regulatory element operably linked to the safety switch gene and a polyadenylated sequence at the 3' end of the hypoimmune gene, or a transcriptional regulatory element operably linked to the hypoimmune gene and a polyadenylated sequence at the 3' end of the safety switch gene.

268. The construct according to any one of claims 261 to 267, wherein the transcriptional control element is selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

269. The construct according to any one of claims 261 to 268, further comprising a vector backbone for lentiviral expression.

270. A method comprising transducing isolated cells with the construct described in claim 269, and selecting the isolated cells that possess the safety switch gene and the low-immunity gene.

271. An isolated cell or population thereof comprising the construct according to any one of claims 261 to 269.

272. The isolated cells or population thereof according to claim 271, wherein the construct is introduced into a target gene locus.

273. The isolated cells or population thereof according to claim 271 or 272, wherein the target gene locus is selected from the group consisting of safe harbor loci selected from the group consisting of AAVS1 locus, CLBYL locus, CXCR4 locus, Rosa26 locus, and CCR5 locus, and immune signaling loci selected from the group consisting of other ligands including B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, RAET1N / ULBP3, and NKG2D.

274. The isolated cells or population thereof according to any one of claims 271 to 273, further comprising the deletion or reduction of expression of MHC class I human leukocyte antigen and / or the deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

275. The isolated cells or population thereof according to any one of claims 271 to 274, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

276. The isolated cells or population thereof according to any one of claims 271 to 275, wherein the isolated cells are low immunogenic and are stem cells.

277. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 276 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, liver cells, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

278. A method for treating a patient requiring cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 277.

279. A method for treating a patient who has been previously administered differentiated cells or a population thereof according to claim 277, the method comprising activating a safety switch in the patient.

280. (1) an essential cell factor, and (2) a recombinant peptide epitope fusion protein containing a peptide epitope exposed on a surface of a different type from the essential cell factor.

281. The protein according to claim 280, wherein the essential cell factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, spliceosome subunit proteins, and their membrane-bound forms.

282. The peptide epitope is from the group consisting of CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD16 epitope, and CD30 epitope. A protein selected from, according to claim 280 or 281.

283. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The protein according to claim 282, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

284. The protein according to any one of claims 280 to 283, wherein the essential cell factor is located at the N-terminus of the fusion protein.

285. The protein according to any one of claims 280 to 284, wherein the peptide epitope is located at the N-terminus of the fusion protein.

286. The protein according to any one of claims 280 to 285, further comprising the essential cell factor and a linker that connects the peptide epitope.

287. The protein according to any one of claims 280 to 286, further comprising a linker located at the N-terminus of the peptide epitope.

288. The protein according to claim 286 or 287, wherein the linker is selected from any one of the linkers provided in Table 3.

289. A construct encoding a recombinant peptide epitope fusion protein, comprising (1) a sequence encoding an essential cell factor, and (2) a sequence encoding a peptide epitope exposed on a surface different from the essential cell factor.

290. The construct according to claim 289, wherein the essential cellular factor is selected from the group consisting of RpS2, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, RpL36, Rpn22, Psmd14, PSMA3, ribosomal subunit proteins, proteasome subunit proteins, spliceosome subunit proteins, and their membrane-bound forms.

291. The peptide epitopes encoded by the sequence of the construct (2) are CD20 epitope, CCR4 epitope, HER2 epitope, CD19 epitope, MUC1 epitope, EGFR epitope, GD2 epitope, PSMA epitope, CD1 A structure according to claim 289 or 290, selected from the group consisting of 6 epitopes and CD30 epitopes.

292. The CD20 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars; the CCR4 epitope is recognized by a therapeutic antibody selected from the group consisting of mogamulizumab and its biosimilars; the HER2 epitope is recognized by a therapeutic antibody selected from the group consisting of margetuximab, trastuzumab, TrasGEX, and their biosimilars; and the CD19 epitope is recognized by a therapeutic antibody selected from the group consisting of obinutuzumab, ubrituximab, okalatuzumab, rituximab, rituximab-RLIb, and their biosimilars. The MUC1 epitope is recognized by a therapeutic antibody selected from the group consisting of MOR208 and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of gatipotuzumab and its biosimilars, the EGFR epitope is recognized by a therapeutic antibody selected from the group consisting of tomzotuximab, RO5083945 (GA201), cetuximab, and their biosimilars, and the GD2 epitope is recognized by Hu14.18K322A, Hu14.18-IL2, Hu3F8, dinutuximab, c. The construct according to claim 291, wherein the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of 60C3-RLIc and its biosimilars, the PSMA epitope is recognized by a therapeutic antibody selected from the group consisting of KM2812 and its biosimilars, the CD30 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of AFM13 and its biosimilars, or the CD20 or CD16 epitope is recognized by a therapeutic antibody selected from the group consisting of (CD20)2xCD16 and its biosimilars.

293. The construct according to any one of claims 289 to 292, wherein the sequence encoding the essential cell factor is located at 5' of the sequence encoding the peptide epitope.

294. The construct according to any one of claims 289 to 293, wherein the sequence encoding the peptide epitope is located at 5' of the sequence encoding the essential cell factor.

295. The construct according to any one of claims 289 to 294, further comprising the sequence encoding the essential cell factor and the sequence encoding the peptide epitope, which is a linker connecting the two sequences.

296. The construct according to any one of claims 289 to 295, further comprising a sequence encoding a linker located at the N-terminus or C-terminus of the fusion protein.

297. The structure according to any one of claims 295 to 296, wherein the linker is selected from any one of the linkers provided in Table 3.

298. The construct according to any one of claims 289 to 297, further comprising a transcriptional regulatory element selected from the group consisting of EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

299. The construct according to any one of claims 289 to 298, further comprising a first homology arm and a second homology arm homologous to a target locus for CRISPR-based homology-directed repair.

300. Claims 289-29 further include a vector backbone for lentiviral expression. A structure as described in any one of item 9.

301. A method comprising transducing isolated cells with the construct described in claim 300, and selecting the isolated cells that express the recombinant peptide epitope fusion protein.

302. Isolated cells or a population thereof comprising a construct according to any one of claims 289 to 300.

303. The isolated cells or population thereof according to claim 302, wherein the isolated cells further comprise deletion or reduced expression of MHC class I human leukocyte antigen and / or deletion or reduced expression of MHC class II human leukocyte antigen compared to unmodified human cells.

304. The isolated cells or population thereof according to any one of claims 302 to 303, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

305. The isolated cells or population thereof according to any one of claims 302 to 304, wherein the isolated cells are low immunogenic and are stem cells.

306. A differentiated cell or population thereof, prepared by culturing the stem cells described in claim 305 under differentiation conditions suitable for the differentiation of the stem cells into cell types selected from the group consisting of cardiac cells, hepatocytes, kidney cells, pancreatic cells, nerve cells, immune cells, mesenchymal cells, and endothelial cells.

307. A method for treating a patient in need of cell therapy, comprising administering to the patient a differentiated cell or population thereof as described in claim 306.

308. A method for treating a patient, comprising administering an antibody that binds to the peptide epitope to a patient who has been previously administered differentiated cells or a population thereof as described in claim 307.

309. The method according to claim 308, wherein the antibody mediates ADCC or CDC.

310. A construct for homology-directed repair into a safe harbor locus, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of the safe harbor locus; (2) a transcriptional regulatory element; (3) an HSVtk safety switch transgene; (4) a sequence encoding a ribosome skipping sequence and / or a linker; (5) a CD47 hypoimmune gene; (6) a polyadenylation sequence; and (7) a second homology arm homologous to a second endogenous sequence of the safe harbor locus.

311. A construct for homology-directed repair into a safe harbor locus, comprising at its 5' to 3' end: (1) a first homology arm homologous to a first endogenous sequence of an immunosignaling locus; (2) a transcriptional regulatory element; (3) an HSVtk safety switch transgene; (4) a sequence encoding a ribosome skipping sequence and / or a linker; (5) a CD47 hypoimmune gene; (6) a polyadenylation sequence; and (7) a second homology arm homologous to a second endogenous sequence of the immunosignaling locus.

312. The transcriptional control element is the EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, The construct according to claim 310 or 311, selected from the group consisting of ACT5C promoter, TRE promoter, CBh promoter, PGK promoter, and UBC promoter.

313. The construct according to any one of claims 310 to 312, further comprising a vector backbone for lentiviral expression.

314. Isolated cells or population thereof comprising a safety switch gene and a hypoimmune gene incorporated within a safe harbor locus or an immune signaling locus, wherein the construct according to any one of claims 310 to 313 is recombined within the endogenous safe harbor locus or the endogenous target locus of the isolated cells.

315. The isolated cells or population thereof according to claim 314, further comprising the deletion or reduction of expression of MHC class I human leukocyte antigen and / or the deletion or reduction of expression of MHC class II human leukocyte antigen compared to unmodified human cells.

316. The isolated cells or population thereof according to claim 314 or 315, further comprising deletion or reduction of expression of CIITA, B2M, and / or NLRC5.

317. The isolated cells or population thereof according to any one of claims 314 to 316, wherein the isolated cells are low immunogenic and are stem cells.

318. A population of differentiated cells or the like, prepared by culturing the stem cells described in claim 317 under differentiation conditions suitable for differentiation into pancreatic cells.

319. The differentiated cells or population thereof according to claim 318, wherein the pancreatic cells are beta islet cells.

320. A method for treating a patient requiring cell therapy, comprising administering to the patient a differentiated cell population or the population described in claim 318 or 319, and activating the safety switch in a patient who has been previously administered the differentiated cell population or the population described in claim 318 or 319.

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