Low immune cells

Abstract

Disclosed herein are compositions and methods relating to isolated cells (e.g., isolated stem cells) that contain disruption of the 3'-UTR of an immunosuppressant, cells differentiated from such stem cells (e.g., pancreatic islet cells or immune cells), and methods of using the cells to treat disease (e.g., diabetes or cancer). Methods of producing (i.e., genetically modifying) the isolated cells (e.g., isolated stem cells) are also provided.

Description

[Technical field]

[0001] Related applications This application claims the benefit under 35 U.S.C. is incorporated herein by reference.

[0002] Reference to electronic sequence listing The contents of the electronic sequence listing (V013870086WO00-SEQ-ZJG.xml; size: 224,720 bytes; and creation date: October 18, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Although not all degenerative diseases can be treated by producing human adult cells derived from stem cells (e.g., human embryonic stem cells or human pluripotent stem cells) for administration to a subject as a cell-based therapy, It offers the possibility of treating most of them. However, the success of such treatments may be limited by the subject's immune response. Strategies that have been considered to overcome immune rejection include reducing or eliminating expression of MHC-1 and / or MHC-II human leukocyte antigens and / or expression of tolerogenic factors in the transplanted cells. This includes increasing the

[0004] Inclusion by reference All publications, patents, and patent applications mentioned herein are the same as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Incorporated herein by reference to this extent. Unless otherwise indicated, the publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety. [Summary of the invention] [Means to solve the problem]

[0005] The present disclosure relates, at least in part, to hypoimmune cells that can be used in cell-based therapies, and methods of producing such hypoimmune cells. In some embodiments, hypoimmune cells are produced from stem cells (eg, human embryonic stem cells or human pluripotent stem cells). As described herein, such stem cells can be engineered to have one or more gene disruptions, for example in the 3'-UTR of genes encoding immunosuppressive factors, producing hypoimmune cells. In the case of stem cells, these can then be further differentiated into the cell type of choice for subsequent use in cell-based therapy. Thus, also provided herein are genetic modifications to cells (eg, stem cells), as well as methods and compositions for performing such genetic modifications. Also provided are methods of using immunocompromised cells to treat diseases (eg, diabetes, cancer).

[0006] Some embodiments of the present disclosure provide isolated cells (e.g., isolated stem cells) comprising a disruption in the 3'-untranslated region (3'-UTR) of an allele encoding an immunosuppressive factor. do. In some embodiments, the disruption comprises a deletion, insertion, translocation, inversion, or substitution in the 3'-UTR. In some embodiments, disruption reduces binding of the 3'-UTR to endogenous RNA binding proteins and / or microRNAs.

[0007] In some embodiments, the immunosuppressive factor is selected from the group consisting of PDL1, CD47, HLA-G, and combinations thereof. In some embodiments, deletions in the 3'-UTR result in increased expression of immunosuppressive factors. In some embodiments, increased expression of the immunosuppressive factor is induced or increased by a cytokine, optionally the cytokine is interferon gamma. In some embodiments, the immunosuppressive factor is PDL1. In some embodiments, the disruption results in deletion of the PDL1 3'-UTR. In some embodiments, the disruption results in an inversion of the PDL1 3'-UTR. In some embodiments, the disruption results in one or more substitutions of nucleotides in the PD-L1 3'-UTR. In some embodiments, the disruption reduces binding of the endogenous microRNA to one or more PDL1 3'-UTRs, and optionally, one or more of the endogenous microRNAs is miR-34a , miR-140, miR-200a, miR-200b / c, miR-142, miR-340, miR-383, miR-424(322), miR-338-5p, miR-324-5p, miR-152, miR-200b, miR-138-5p, miR-195, miR-16, miR-15a, miR15b miR-193a-3p, miR-497-5p, miR-33a, miR17-5p, miR-155, and miR- selected from the group consisting of 513. In some embodiments, the disruption includes any of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 45, 48, 36, 58, 59, 61, 63, 65, 67, 69, 71, and 73. resulting in a deletion of 1 to 7 nucleotides in one or more of the PDL1 3'-UTR sequences described in one or more of the PDL1 3'-UTR sequences. In some embodiments, the disruption is to any one of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 44, 47, 57, 60, 62, 64, 66, 68, 70, and 72. Results in deletions of 1-24 nucleotides in one or more of the PDL1 3'-UTR sequences described.

[0008] In some embodiments, the immunosuppressive factor is HLA-G. In some embodiments, the disruption reduces binding of the endogenous microRNA to one or more HLA-G 3'-UTRs, and optionally, one or more of the endogenous microRNAs -133A, miR-148A, miR-148B, miR-152, miR-548q and / or miR-628-5p. In some embodiments, the disruption is a deletion of at least 5 contiguous nucleotides starting at and including position +2961 of the HLA-G 3'-UTR, and / or a deletion of at least 5 contiguous nucleotides at position +2961. results in the insertion of nucleotides. In some embodiments, the disruption is in the HLA-G 3'-UTR sequence set forth in SEQ ID NO:74. In some embodiments, the disruption results in the deletion of at least one nucleotide of the HLA-G 3'-UTR sequence set forth in SEQ ID NO:75. In some embodiments, the disruption results in one or more mutations selected from C120G, G252C, A297G, and / or C306G of the HLA-G 3'-UTR sequence set forth in SEQ ID NO:74.

[0009] In some embodiments, the isolated cell (e.g., isolated stem cell) has CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA- E, further insertion of sequences encoding HLA-G, C1 inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9 include. In some embodiments, insertion of a sequence encoding CD47 into the PDL1 3'-UTR locus results in an RNA comprising an immunosuppressive factor and a sequence encoding CD47.

[0010] In some embodiments, the isolated cells (e.g., isolated stem cells) contain CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL -35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9 encoding sequences into the safe harbor locus.

[0011] In some embodiments, the isolated cell (eg, isolated stem cell) does not contain insertions of foreign coding sequences into its genome. In some embodiments, the isolated cells (e.g., isolated stem cells) exhibit increased expression of MHC-I and MHC-II human leukocyte antigens (HLA) compared to wild-type stem cells of the same cell type. is decreasing. In some embodiments, the reduced expression of MHC-I HLA is due to a disruption in the allele encoding β-2 microglobulin (B2M). In some embodiments, the reduced expression of MHC-II HLA is due to a disruption in an allele encoding class II major histocompatibility complex transactivator (CIITA).

[0012] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are human stem cells. In some embodiments, the stem cells are negative for A antigen and negative for B antigen. In some embodiments, the stem cells are negative for A antigen. In some embodiments, the stem cells are negative for B antigen. In some embodiments, the stem cells are negative for A antigen and positive for B antigen. In some embodiments, the stem cells are positive for A antigen and negative for B antigen. In some embodiments, the stem cells are negative for Rh antigen.

[0013] Other aspects of the disclosure provide cells differentiated from any of the isolated stem cells described herein. In some embodiments, the cells are fibroblasts, endothelial cells, definitive endoderm cells, gastrula cells, pancreatic progenitor cells, pancreatic endocrine cells, pancreatic islet cells, stem cell-derived beta cells, stem cell-derived alpha cells, stem cell-derived delta cells, stem cell-derived enterochromaffin (EC) cells, insulin-producing cells, insulin-positive beta-like cells, hematopoietic stem cells, hematopoietic progenitor cells, muscle cells, satellite stem cells, hepatocytes, neurons, or immune cells. . In some embodiments, the cell is an immune cell, and optionally the immune cell expresses a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the cells are less immunogenic compared to cells of the same cell type.

[0014] Compositions comprising isolated cells (eg, isolated stem cells) or cells differentiated from isolated stem cells described herein are also provided. In some embodiments, the composition comprises NKX6.1-positive, ISL-positive cells and NKX6.1-negative, ISL-positive cells, and the population has more NKX6.1-positive than NKX6.1-negative, ISL-positive cells. , including ISL-positive cells, at least 15% of the cells in the population are NKX6.1-negative, ISL-positive cells, and less than 12% of the cells in the population are NKX6.1-negative, ISL-negative cells.

[0015] Additionally, a method comprising administering an isolated cell (e.g., an isolated stem cell) or a cell differentiated from an isolated stem cell described herein to a subject in need thereof. Provided herein. In some embodiments, the method is a method of treating diabetes, comprising producing pancreatic islet cells differentiated from isolated stem cells described herein, or compositions comprising such cells. and administering to a subject in need thereof. In some embodiments, the method is a method of treating cancer, wherein the method comprises administering an immune cell differentiated from an isolated stem cell described herein, or a composition comprising such a cell. to a subject in need thereof. In some embodiments, the cancer is a blood cancer.

[0016] Another aspect of the disclosure is a method of producing an isolated cell (e.g., an isolated stem cell) described herein, comprising: an RNA targeting endonuclease and an allele encoding an immunosuppressive factor; A method is provided comprising delivering to a stem cell a CRISPR system comprising one or more guide RNAs (gRNAs) comprising a nucleotide sequence that targets the 3'-UTR of a gene.

[0017] In some embodiments, the RNA targeting endonuclease is a Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a Cas12i protein, or a Casφ protein. In some embodiments, the immunosuppressive factor is PDL1, CD47, or HLA-G. In some embodiments, the immunosuppressive factor is PDL1. In some embodiments, the gRNA targets a target sequence corresponding to positions 1003-1022 or 1021-1040 of the PDL1 sequence set forth in SEQ ID NO: 1, or the 3'- Target the target sequence downstream of the UTR. In some embodiments, the composition comprises a first gRNA targeting a target sequence corresponding to positions 1003 to 1022 or positions 1021 to 1040 of the PDL1 sequence set forth in SEQ ID NO: 1, and a PDL1 on the opposite strand. a second gRNA that targets a target sequence downstream of the 3'-UTR of the gRNA. In some embodiments, the gRNA is modified. In some embodiments, gRNA is delivered in lipid nanoparticles (LNPs). In some embodiments, the gRNA is delivered via a nucleic acid that includes a nucleotide sequence encoding the gRNA, and optionally the nucleic acid is a viral vector. In some embodiments, the RNA targeting endonuclease is delivered via a nucleic acid that includes a nucleotide sequence encoding the RNA targeting endonuclease, optionally the nucleic acid is a viral vector.

[0018] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will become apparent from the following drawings and detailed description of some embodiments, and from the appended claims.

[0019] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For clarity, not all components are shown in all figures. [Brief explanation of the drawing]

[0020] [Figure 1] Figures 1A and 1B are a series of graphs showing that CD69 expression, which indicates T cell activation, is reduced by disrupting the 3'-UTR of PDL1. Figure 1A shows media alone, media containing CD3 / CD28, endothelial cells differentiated from wild-type human embryonic stem cells (hESCs), endothelial cells differentiated from hESCs with B2M / CIITA double knockout (DKO), or endothelial cells differentiated from hESCs with PDL1. Graph showing the percentage of CD8+ T cells with surface expression of CD69 when mixed with endothelial cells differentiated from hESCs of three 3'-UTR deleted clones (#12, #19, #25) It is. FIG. 1B is a graph showing HLA-I expression measured by flow cytometry and reported as mean fluorescence intensity (MFI) for the indicated cells. Cells in which the PDL1 3'-UTR is disrupted express HLA-I at levels comparable to wild-type cells. [Figure 2] Figure 2 shows media alone, the positive control GMP-WCB, three clones of endothelial cells differentiated from wild-type human embryonic stem cells (hESCs), or three clones of hESCs with a deletion in the 3'-UTR of PDL1. FIG. 3 is a graph showing the percentage of CD8+ T cells with surface expression of CD69 when mixed with endothelial cells differentiated from . *** indicates p<0.001. Consistent with what was observed in Figure 1A, deletion of the 3'-UTR of PDL1 reduced T cell activation to an extent similar to that observed with ablation of HLA classes I and II. [Figure 3] Figure 3 shows endothelial cells differentiated from wild-type human embryonic stem cells (hESCs), or endothelial cells differentiated without stimulation from hESCs in which the 3'-UTR of PDL1 was deleted, or Figure 2 is a graph showing the surface expression of PDL1 (measured by flow cytometry and reported as MFI) in CD8+ T cells or endothelial cells differentiated in the presence of IFNγ from deleted hESCs. [Figure 4] FIG. 4 is a graph showing the percentage of endothelial cells remaining (% survival) after co-culture with purified human CD8+ T cells. The cells tested were from wild-type human embryonic stem cells (hESCs), four different clones of hESCs with a B2M / CIITA double knockout (DKO), or four different clones of hESCs with a deletion of the 3'-UTR of PDL1. differentiated. The results show that deletion of the 3'-UTR of PDL1 renders cells resistant to T cell-mediated killing. [Details for carrying out the invention]

[0021] The following description and examples describe embodiments of the disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein, as such may vary. Those skilled in the art will recognize that there are numerous variations and modifications to this disclosure that fall within the scope of this disclosure.

[0022] All terms are intended to be understood as understood by one of ordinary skill in the art. 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 disclosure pertains.

[0023] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided individually or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure can also be practiced in a single embodiment.

[0024] The following definitions supplement definitions in the art and are directed to this application and are not attributed to any related or unrelated examples, such as any widely shared patents or patent applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0025] The use of the singular in this application includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0026] The use of "or" in this application means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases, "A, B, and / or C" or "A, B, C, or any combination thereof" are interchangeable with "Individually A, Individually B, Individually C." , A and B, B and C, A and C, and A, B, and C. The term "or" may be used either conjunctive or disjunctive, unless the context specifically indicates otherwise disjunctive use.

[0027] Furthermore, the use of the term "including" and other forms such as "include, includes" and "included" is not limiting. References in the specification to "some embodiments," "an embodiment, one embodiment," or "other embodiments" refer to specific features described in connection with that embodiment. , structure, or characteristic is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0028] As used in this specification and the claims, the words "comprising" (and any forms of comprising such as "comprise" and "comprises"), "having" (and "have" and " "including" (and any forms of including such as "includes" and "include"), or "containing" (as well as "contains" and "contain Any form of containing (such as ``containing'') is inclusive or open-ended and does not exclude additional elements or method steps not mentioned. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the present disclosure, and vice versa. Additionally, the compositions of the present disclosure can be used to accomplish the methods of the present disclosure.

[0029] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, and this depends in part on how that value was measured or determined; For example, this may be due to limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as is common practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, an amount of "about 10" includes any amount from 10 and 9 to 11. In yet another example, the term "about" in connection with a reference numerical value means plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or It may also include a range of 1%. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5 times, more preferably within 2 times, of a certain value. Where specific values ​​are stated in applications and claims, the term "about" should be assumed to mean within the permissible error range for the specific value, unless stated otherwise. be.

[0030] As used herein, the term "diabetes" and its grammatical equivalents may refer to a disease characterized by high blood sugar levels over an extended period of time. For example, as used herein, the term "diabetes" and its grammatical equivalents include, but are not limited to, type 1 diabetes, type 2 diabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes, and mitochondrial diabetes. can refer to all or any types of diabetes including. In some instances, diabetes may be a form of hereditary diabetes. In some embodiments, diabetes can be an autoimmune type of diabetes.

[0031] The term "endocrine cell", unless otherwise specified, refers to the cells of the living pancreas, such as "islet", "islet cell", "islet equivalent", "islet-like cell", "pancreatic islet", and grammatical equivalents thereof. can refer to hormone-producing cells present in In one embodiment, endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Pancreatic islet cells can include various types of cells including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cell can also refer to a group of cells, a cluster of cells, etc.

[0032] The terms "progenitor cell" and "precursor" cell are used interchangeably herein to refer to a cell that is more primitive (e.g., a cell that is fully differentiated in a developmental pathway or development) compared to a cell that can arise by differentiation. refers to cells with a cellular phenotype (at an earlier step than Progenitor cells can also often have significant or extremely high proliferative potential. Progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0033] The term "progenitors thereof" as used in reference to insulin-positive endocrine cells refers to cells that have differentiated into insulin-positive endocrine cells, including, for example, pluripotent stem cells, definitive endoderm cells, gastrula cells, pancreatic progenitor cells, or endocrine progenitor cells. It can refer to any cell that can be obtained, and if cultured under appropriate conditions, the precursor cell can be differentiated into an insulin-positive endocrine cell.

[0034] The terms "stem cell-derived beta cells," "SC-beta cells," "functional beta cells," "functional pancreatic beta cells," "mature SC-beta cells," and their grammatical equivalents refer to pancreatic beta cells. cells (e.g., , non-natural pancreatic β cells). In some embodiments, the terms "SC-β cell" and "non-natural β cell" as used herein are interchangeable. In some embodiments, "SC-β cells" express lower levels of MAFA than pancreatic β cells from healthy adult human patients. In some embodiments, "SC-β cells" express higher levels of MAFB than pancreatic β cells from healthy adult human patients. In some embodiments, "SC-β cells" express higher levels of SIX2, HOPX, IAPP and / or UCN3 than pancreatic β cells from healthy adult human patients. In some embodiments, "SC-β cells" include mature pancreatic cells. Since the methods of the present disclosure can use any cell as a starting point to derive SC-β cells from any insulin-positive endocrine cell or its precursor, SC-β cells can be derived from stem cells ( It should be understood that progenitor cells such as embryonic stem cells, induced pluripotent stem cells, definitive endoderm cells, partially reprogrammed somatic cells (induced a somatic cell that has been partially reprogrammed to a state intermediate between a pluripotent stem cell and the somatic cell from which it is derived), a pluripotent cell, a totipotent cell, a transdifferentiated version of any of the aforementioned cells, etc. (the invention is not intended to be so limited). In some embodiments, the SC-β cells exhibit a response to multiple glucose challenges (eg, at least one, at least two, or a series of at least three or more glucose challenges). In some embodiments, the response is similar to the response of endogenous pancreatic islets (eg, human pancreatic islets) to multiple glucose challenges. In some embodiments, the SC-β cell morphology is similar to endogenous β cell morphology. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to those of endogenous β cells. In some embodiments, the GSIS response of SC-β cells can be observed within 2 weeks after transplanting the SC-β cells into a host (eg, human or animal). In some embodiments, the GSIS response of SC-β cells can be observed within 3 weeks after transplanting the SC-β cells into a host (eg, human or animal). In some embodiments, the GSIS response of SC-β cells can be observed within 4 weeks after transplanting the SC-β cells into a host (eg, human or animal). In some embodiments, the GSIS response of SC-β cells can be observed within 1-3 months after transplantation of the SC-β cells into a host (eg, human or animal). In some embodiments, SC-β cells package insulin into secretory granules. In some embodiments, the SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic agents (eg, secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not aberrantly co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptides. In some embodiments, SC-β cells exhibit low replication rates. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose. In some embodiments, the stimulation index of a cell is characterized by the ratio of insulin secreted in response to a high glucose concentration (eg, 15 mM) compared to a low glucose concentration (eg, 2.5 mM).

[0035] The terms “stem cell-derived α cells,” “SC-α cells,” “functional α cells,” “functional pancreatic α cells,” “mature SC-α cells,” and their grammatical equivalents refer to pancreatic α cells. cells that express glucagon and secrete functional glucagon (e.g., non-native pancreatic alpha cells). can mean In some embodiments, the "SC-α cells" do not express somatostatin. In some embodiments, the "SC-α cells" do not express insulin. In some embodiments, the terms "SC-α cell" and "non-natural α cell" as used herein are interchangeable. In some embodiments, "SC-α cells" include mature pancreatic cells.

[0036] The terms "stem cell-derived delta cells," "SC-delta cells," "functional delta cells," "functional pancreatic delta cells," "mature SC-delta cells," and their grammatical equivalents refer to pancreatic delta cells. can refer to cells (eg, non-natural pancreatic delta cells) that display, express, and secrete at least one marker (eg, somatostatin) indicative of somatostatin. In some embodiments, the "SC-δ cells" do not express glucagon. In some embodiments, the "SC-δ cells" do not express insulin. In some embodiments, the terms "SC-δ cell" and "non-native δ cell" as used herein are interchangeable. In some embodiments, "SC-δ cells" include mature pancreatic cells.

[0037] The terms "stem cell-derived enterochromaffin (EC) cells", "SC-EC cells", and their grammatical equivalents are defined as having at least one marker indicative of pancreatic EC cells (e.g., VMAT1 (vesicular monoamine transporter 1)). ), expressing NKX6.1 but not ISL1) (eg, non-native pancreatic EC cells). In some embodiments, the terms "SC-EC cell" and "non-native EC cell" as used herein are interchangeable.

[0038] Similar to SC-β cells, SC-α, SC-δ, and SC-EC cells do not need to be derived (e.g., directly) from stem cells, as the disclosed method uses SC- It should be understood that this is because SC-α cells can be derived from other progenitor cells produced during in vitro differentiation of β-cells (e.g., the present invention is not limited in this way). embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., an intermediate state between an induced pluripotent stem cell and the somatic cell from which it is derived). (somatic cells that have been partially reprogrammed), pluripotent cells, totipotent cells, transdifferentiated versions of any of the aforementioned cells, etc.) can be used.

[0039] As used herein, the term "insulinogenic cell" and its grammatical equivalents refers to a cell that differentiates from a pancreatic progenitor cell or its precursor and secretes insulin. Insulin-producing cells, as that term is used herein, are pancreatic beta cells, as well as those that synthesize insulin in a constitutive or inducible manner (e.g., transcribe the insulin gene, translate proinsulin mRNA, and pancreatic β-like cells (e.g., insulin that modifies proinsulin mRNA into insulin protein), expresses (e.g., achieves the phenotypic trait conveyed by the insulin gene), or secretes (releases insulin into the extracellular space) positive endocrine cells). The population of insulin-producing cells, e.g., produced by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present disclosure, comprises at least one pancreatic β-cell or β-like cell, e.g., an endogenous β-cell. or at least two characteristics, and may exhibit a glucose-stimulated insulin secretion (GSIS) response similar to endogenous mature beta cells. For example, a population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic beta cells or SC-beta cells, as well as non-insulin producing cells (e.g., other than not producing or secreting insulin). cells with a cell-like phenotype).

[0040] The terms "insulin-positive beta-like cells", "insulin-positive endocrine cells", and their grammatical equivalents exhibit at least one marker indicative of pancreatic beta cells and also express insulin, but are characteristic of endogenous beta cells. can refer to cells that lack a typical glucose-stimulated insulin secretion (GSIS) response (eg, pancreatic endocrine cells). Exemplary markers of "insulin positive endocrine cells" include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Islet1), and insulin.

[0041] The term "beta cell marker" refers to, without limitation, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analyzes expressed or present in pancreatic beta cells. means substance. Exemplary beta cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2 , GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3 beta, VMAT2, NKX6.1, and MafA, and Zhang et al., Diabetes. 50(10):2231 -6 (2001). In some embodiments, the beta-cell marker is a nuclear beta-cell marker. In some embodiments, the beta cell marker is PDX1 or PH3.

[0042] The term "pancreatic endocrine marker" refers to, without limitation, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analyzes expressed or present in pancreatic endocrine cells. It can mean matter. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0043] The terms "pancreatic progenitor cell", "pancreatic endocrine progenitor cell", "pancreatic progenitor", "pancreatic endocrine precursor", and their grammatical equivalents are used interchangeably herein and refer to pancreatic endocrine cell, pancreatic endocrine cell, It can refer to a stem cell capable of becoming a pancreatic exocrine cell, or a pancreatic hormone expressing cell capable of forming a pancreatic ductal cell. These cells include at least one type of pancreatic cell, such as beta cells that produce insulin, alpha cells that produce glucagon, delta cells (or D cells) that produce somatostatin, and / or F cells that produce pancreatic polypeptides. It is involved in differentiation towards. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0044] As used herein, the term "PDX1-positive pancreatic progenitor cells" may refer to cells that are pancreatic endoderm (PE) cells that have the ability to differentiate into SC-β cells, such as pancreatic β cells. PDX1-positive pancreatic progenitor cells express the marker PDX1. Other markers include, but are not limited to, Cdcp1, or Ptfla, or HNF6 or NRx2.2. Expression of PDX1 can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-PDX1 antibodies or quantitative RT-PCR. In some cases, PDX1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, a PDX1-positive pancreatic progenitor cell can also be referred to as a PDX1-positive, NKX6.1-negative pancreatic progenitor cell, since it lacks expression of NKX6.1. In some cases, PDX1-positive pancreatic progenitor cells can also be termed "pancreatic foregut endoderm cells."

[0045] The terms "PDX1-positive, NKX6.1-positive pancreatic progenitor cells" and "NKX6.1-positive pancreatic progenitor cells" are used interchangeably herein and refer to the ability to differentiate into insulin-producing cells such as pancreatic beta cells. can refer to cells that are pancreatic endoderm (PE) cells with PDX1-positive and NKX6-1-positive pancreatic progenitor cells express the markers PDX1 and NKX6-1. Other markers may include, but are not limited to, Cdcp1, Ptf1a, HNF6 or NRx2.2. Expression of NKX6-1 can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-NKX6-1 antibodies or quantitative RT-PCR. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some cases, PDX1-positive, NKX6-1-positive pancreatic progenitor cells can also be named "pancreatic foregut progenitor cells."

[0046] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify a protein and the gene encoding it that is expressed in pancreatic endocrine progenitor cells. The term "differentiated cell" or its grammatical equivalent, as defined herein, means any primary cell that is not pluripotent in nature. Alternatively, the term "differentiated cell" refers to a cell of a highly specialized cell type that is derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cell differentiation process. It is possible. Without wishing to be limited by theory, pluripotent stem cells during normal ontogeny can initially differentiate into endodermal cells that can form pancreatic cells and other endodermal cell types. . Further differentiation of endodermal cells leads to the pancreatic pathway, where about 98% of the cells become exocrine, tubular, or matrix cells and about 2% endocrine cells. Early endocrine cells are pancreatic islet progenitor cells, which can then further differentiate into insulin-producing cells (eg, functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptides. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0047] As used herein, the term "somatic cell" may mean any cell that forms a living organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are the sperm and egg that fuse during fertilization to produce a cell called a zygote, which is the entire mammalian embryo. occurs. Apart from sperm and eggs (gametocytes) and undifferentiated stem cells, which are the cells from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bones, blood, and connective tissue are all made from body cells. In some embodiments, the somatic cells are "non-embryonic somatic cells," meaning somatic cells that are not present in or obtained from the embryo and do not result from in vitro propagation of such cells. . In some embodiments, the somatic cell is an "adult somatic cell," meaning a cell that resides in or is obtained from an organism other than an embryo or fetus, or results from in vitro propagation of such a cell. do. Unless otherwise indicated, methods for converting at least one insulin-positive endocrine cell or its precursor into an insulin-producing glucose-responsive cell may be performed either in vivo and in vitro, or both in vivo and in vitro. (in vivo is carried out when at least one insulin-positive endocrine cell or its precursor is present in the subject; in vitro is carried out when at least one isolated insulin-positive endocrine cell or its precursor is maintained in culture) (performed using insulin-positive endocrine cells or their precursors).

[0048] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development. As used herein, the term "endodermal cell" may mean a cell that is from one of the three primary germ cell layers in a very early embryo (the other two germ cell layers are are mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate, first to give rise to the fetal intestine, then to the lining of the respiratory and gastrointestinal tract (such as the intestines), liver, and pancreas.

[0049] As used herein, the term "cell of endodermal origin" can mean any cell that has developed or differentiated from an endodermal cell. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, liver and pancreatic progenitor cells (also referred to as pancreatic progenitor cells) develop from endodermal cells of the embryonic foregut. Once they are identified, liver and pancreatic progenitor cells rapidly acquire distinct cellular functions and regenerative abilities. These changes are driven by inducing signals and genetic regulators that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the strong need for hepatocytes and pancreatic β cells in therapeutic treatments for liver failure and type I diabetes. Studies in diverse model organisms and humans have provided guidance on how to induce hepatocyte and pancreatic cell differentiation and promote hepatocyte and beta cell differentiation from diverse stem and progenitor cell types. A network of evolutionarily conserved inducing signals and transcription factors has been uncovered.

[0050] As used herein, the term "definitive endoderm" can refer to cells that can differentiate from endodermal cells and differentiate into SC-β cells (eg, pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers characteristic of definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3B, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, Goosecoid, C-Kit, CD99, CMKOR1 , and CRIP1. In particular, the definitive endoderm cells herein express Sox17 and, in some embodiments, Sox17 and HNF3B, and do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker PDX1 (eg they are PDX1 negative). Definitive endoderm cells have the ability to differentiate into cells including liver, lung, pancreas, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other markers of definitive endoderm can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0051] The term "pancreatic endoderm" can refer to cells of endodermal origin that are capable of differentiating into multiple pancreatic lineages, including pancreatic beta cells, but no longer have the ability to differentiate into non-pancreatic lineages. The term "islet cell" refers to a cell population that includes different types of pancreatic endocrine cells (β cells, α cells, δ cells, ε cells) and enterochromaffin (EC) cells, e.g. Xavier et al. (J Clin Med. 2018 Mar; 7(3): 54), incorporated herein by reference.

[0052] As used herein, the term "gastrulocyte" or "intestinal cell" refers to a cell that differentiates from endodermal cells and is capable of differentiating into SC-β cells (e.g., pancreatic β cells). I can do it. Gastrocytes express at least one of the following markers: HNP1-β, HNF3-β, or HNF4-α. In some cases, the gastrula cells are FOXA2 positive and SOX2 positive, ie, express both FOXA2 (also known as HNF3-β) and SOX2. In some cases, gastrula cells are FOXA2 positive and PDX1 negative, ie, express FOXA2 but not PDX1. Gastrocytes have the ability to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. Expression of HNF1-β and other gastrula markers can be assessed by any method known to those skilled in the art, such as, for example, immunochemistry using anti-HNF1-β antibodies.

[0053] As used herein, the term "stem cell" means to give rise to more progenitor cells that have the ability to proliferate and produce a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells. Can refer to undifferentiated cells. retaining one or more cells with the developmental potential of the parent while the daughter cells themselves are induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types I can do it. The term "stem cell" refers to cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances, and that under certain circumstances retain the ability to proliferate without substantially differentiating. can refer to a subset of progenitor cells that are In one embodiment, the term stem cell generally means that its descendants (progeny) acquire completely distinct characteristics, e.g., by differentiation, as occurs in the gradual diversification of embryonic cells and tissues. refers to a naturally occurring mother cell that often specializes in different directions. Cell differentiation is a complex process that typically occurs through many cell divisions. The differentiated cells may be derived from pluripotent cells, the pluripotent cells themselves being derived from pluripotent cells, and so on. Although each of these pluripotent cells can be considered a stem cell, the range of cell types that each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such ability may be natural or may be artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can generate progeny with two or more different cell types, but this is not required for "stemness." "Self-renewal" is another classic part of the definition of stem cells, which is important as used herein. In theory, self-replication can occur by either of two main mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem cell state and other daughters expressing several different other specific functions and phenotypes. Alternatively, some of the stem cells in the population can divide symmetrically into two stem cells, whereby some stem cells in the population are maintained as a whole while other cells in the population have differentiated progeny. produce only Formally, a cell that begins as a stem cell progresses toward a differentiated phenotype, but it is also possible to later "reverse" and reverse express the stem cell phenotype, and this is well understood by those skilled in the art. is a term often referred to as "dedifferentiation" or "reprogramming" or "reverse differentiation". As used herein, the term "pluripotent stem cell" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0054] As used herein, the term "pluripotent" refers to the ability to differentiate into two or more differentiated cell types under a variety of conditions, preferably into cell types characteristic of all three germinal cell layers. can refer to cells that have Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably all three germ layers, using, for example, a nude mouse teratoma formation assay. Although pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not, by itself, render these cells pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as that term is defined herein) generally have the ability to divide only a limited number of times in culture, compared to primary parental cells. It is also characterized by the ability to be passaged for extended periods without losing growth potential.

[0055] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and mean that cells can be rendered non-pluripotent by, for example, inducing forced expression of one or more genes. Can refer to pluripotent stem cells that are artificially derived (eg, induced or by complete reversal) from cells, typically adult somatic cells.

[0056] The term "phenotype" can mean any one or several biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype. The terms "patient," "subject," and "individual" are used interchangeably and can mean either a human or a non-human animal. "Non-human animal" and "non-human mammal," as used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. is included. The term "subject" also includes any vertebrate including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or another mammal, such as, for example, a dog, cat, horse, other domestic animal, or cow, sheep, pig, or other production mammal. A “patient in need” or “subject in need” herein refers to a patient diagnosed with or suspected of having, for example, a disease or disorder, not limited to diabetes.

[0057] As used herein, "administering" can mean providing one or more compositions described herein to a patient or subject. By way of example and without limitation, administration of the composition, such as injection, may include intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. It can be carried out by One or more such routes can be taken. Parenteral administration may be, for example, by bolus injection or gradual perfusion over time. Alternatively, or simultaneously, administration may be by the oral route. Additionally, administration may involve surgically depositing a bolus or pellet of cells or positioning a medical device. In one embodiment, a composition of the present disclosure comprises an effective amount of a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein to treat or prevent a proliferative disorder. may include engineered cells or host cells that express . Pharmaceutical compositions may include the cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants. agents, chelating agents such as EDTA or glutathione, adjuvants (eg, aluminum hydroxide), and preservatives.

[0058] Some numerical values ​​disclosed throughout are referred to, for example, "X is at least or at least about 100, or 200 [or any number]." This number includes the number itself and i) X is at least 100; ii) X is at least 200; iii) X is at least about 100, and iv) X is at least about 200 All of the following are included.

[0059] All these different combinations are contemplated by the numerical values ​​disclosed throughout. Unless specifically indicated otherwise, all numerical values ​​disclosed, whether for administration of therapeutic agents, days, months, years, weights, doses, etc., shall be treated as such. It should be interpreted as follows.

[0060] Ranges disclosed throughout may include, for example, ``X is administered on or about days 1-2, or on or about days 2-3 [or any numerical range]. ” is sometimes called. This range includes the number itself (e.g. the endpoint of the range) and i) X is administered between days 1 and 2; ii) X is administered between days 2 and 3; iii) X is administered between about days 1 and 2; iv) X is administered between about days 2 and 3; v) X is administered between day 1 and about day 2; vi) X is administered between day 2 and about day 3; vii) X is administered between about day 1 and about day 2, and viii) X is administered between about day 2 and about day 3 All of the following are included.

[0061] All these different combinations are contemplated by the generally disclosed ranges. Unless specifically indicated otherwise, all ranges disclosed, whether as to administration of a therapeutic agent or as to days, months, years, weights, doses, etc. It should be interpreted as follows.

[0062] This disclosure contemplates the complements (eg, reverse complements) and / or RNA equivalents of any of the DNA sequences disclosed herein. For example, any of the DNA sequences disclosed herein can be presented alternatively, replacing each "T" in the sequence with a "U" to generate an RNA equivalent.

[0063] low immune cells The present disclosure, in some aspects, provides cells that are hypoimmune. In some embodiments, the present disclosure provides isolated cells (eg, somatic cells) that are hypoimmune. In some embodiments, the present disclosure provides stem cells that can be differentiated into cells (eg, somatic cells) that are hypoimmune. Such differentiated cells (eg, somatic cells) can be used, in some embodiments, to be administered to a subject to treat a disease (eg, diabetes or cancer). In some embodiments, a cell described herein (e.g., an isolated cell, or a cell differentiated from an isolated stem cell) is compared to a wild type cell of the same type. have low immunogenicity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less immunogenic). In some embodiments, a cell described herein (e.g., an isolated cell, or a cell differentiated from an isolated stem cell) is compared to a wild type cell of the same type. reduced level of CD8+ T cell activation (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) %, at least 95%, or at least 99%). In some embodiments, a cell described herein (e.g., an isolated cell, or a cell differentiated from an isolated stem cell) is compared to a wild type cell of the same type. increased resistance (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold or more).

[0064] In some embodiments, a cell of the present disclosure (e.g., an isolated cell or a cell differentiated from an isolated stem cell) comprises a 3'-untranslated region (3'-untranslated region) of an allele encoding an immunosuppressive factor. '-UTR). In some embodiments, disruptions in the 3'-UTR include deletions (e.g., fragments of the 3'-UTR or deletion of the entire 3'-UTR), insertions, translocations, inversions (e.g., 3'- or substitutions (eg, substitutions of one or more nucleotides within the 3'-UTR), or combinations thereof. Thus, in some embodiments, disruption of the 3'-UTR comprises an insertion. In some embodiments, disruption of the 3'-UTR comprises an insertion. In some embodiments, disruption of the 3'-UTR comprises a translocation. In some embodiments, disruption of the 3'-UTR comprises an inversion. In some embodiments, disruption of the 3'-UTR comprises a substitution. In some embodiments, any genetic modification described herein is a homozygous modification. In some embodiments, the genetic modifications described herein are heterozygous modifications.

[0065] In some embodiments, the 3'-UTR is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, Contains nucleotide sequences that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. For example, in some embodiments, the 3'-UTR is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, Contains nucleotide sequences that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0066] In some embodiments, the present disclosure contemplates cells in which sequences within the 3'-UTR of an immunosuppressive factor have been disrupted. In some embodiments, the disruption includes 1, 2, 3, 4, 5, 1-25, 1-20, 1-15, 1-10, 1-5, 1~3, 5~25, 5~20, 5~15, 5~10, 10~25, 10~20, 10~15, 15~25, 15~20, 20~25, 25~100, 100~ Contains deletions of 200, 200-300, 300-400, 400-500, 500-1000, or 1000-5000 nucleotides. In some embodiments, the disruption is 1, 2, 3, 4, 5, 1-25, 1-20, 1-15, 1-10, 1-5, Contains insertions of 1-3, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or 20-25 nucleotides. In some embodiments, the disruption is 1, 2, 3, 4, 5, 1-25, 1-20, 1-15, 1-10, 1-5, 1 in the 3'-UTR of the immunosuppressive factor. Contains substitutions of ~3, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or 20-25 nucleotides. In certain embodiments, disruption of the 3'-UTR results in reduced or eliminated binding of miRNA to the 3'-UTR. In certain embodiments, disruption of the 3'-UTR results in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 of the binding of the miRNA to the 3'-UTR. %, 95%, or 100% reduction. In certain embodiments, disruption of the 3'-UTR results in at least 10%, 30%, 50%, 75%, 100% of the expression of the immunosuppressive factor compared to cells in which the 3'-UTR is not disrupted. , resulting in an increase of 150%, 200%, or 250%. In some embodiments, the disrupted sequence comprises the sequence ATTTA, ATTTTA, or ATTTTTA. In some embodiments, the disrupted sequence is 1-25, 1-20, 1-15, 1-10, 1-5 from the sequence ATTTA, ATTTTA, or ATTTTTA in the 3'-UTR of the immunosuppressive factor. , 1-3, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or within 20-25 nucleotides.

[0067] In some embodiments, the present disclosure provides that endogenous RNA binding proteins and / or microRNAs within a cell are unable to bind or have significantly reduced binding to the 3'-UTR of RNA encoded by an immunosuppressive gene. As such, cells in which the 3'-UTR of intracellular immunosuppressive genes are disrupted are considered. In some embodiments, the RNA binding protein and / or microRNA is unable to bind to the 3'-UTR due to a deletion of one or more nucleotides in the binding site of the 3'-UTR to the microRNA. . In some embodiments, the RNA binding protein and / or microRNA has one or more nucleotide insertions in the binding site of the 3'-UTR for the microRNA (e.g., several nucleotides have been inserted). (or the entire transgene is inserted into the binding site for the microRNA of an immunosuppressive gene), it cannot bind to the 3'-UTR. In some embodiments, the RNA binding protein and / or the microRNA binds the microRNA so that the microRNA is no longer able to bind to the 3'-UTR (e.g., breaking complementarity / base pairing). It is unable to bind to the 3'-UTR because one or more nucleotides in the binding site of the 3'-UTR to RNA have been substituted. In some embodiments, the immunosuppressive gene is PDL1 and the microRNA is miR-34a, miR-140, miR-200a, miR-200b / c, miR-142, miR-340, miR-383, miR -424(322), miR-338-5p, miR-324-5p, miR-152, miR-200b, miR-138-5p, miR-195, miR-16, miR-15, miR-193a-3p, Any one or more of miR-497-5p, miR-33a, miR17-5p, miR-155 and / or miR-513. For example, Xie et al., 2017 PLOS One, DOI:10.1371 / journal.pone.0168822; Zhao et al., 2016, Oncotarget, 7(29):45370-84; He et al., 2018 Biomedicine and Pharmacology, 98:95 ~101 pages; Tao et al., 2018, Cell Physiol Biochem., 48:801~814; Kao et al., 2017, J. Thoracic Oncology, 12(9):1421~1433; Audrito et al., 2017, Oncotarget , 8(9):15894~15911;Holla et al., 2016, Scientific Reports, 6(24193);Danbaran et al., 2020, International Immunopharmacology, 84:106594;Gong et al., 2009, J Immunol., 182(3) :1325~1333; Chen et al., 2014, Nat. Commun., 5:5241; Wang et al., 2015, Cellular Signaling, 27(3):443~452; Xu et al., 2016, Nat. Comm. ., 7:11406, and Dong et al., 2018, Oncogene, 37:5257-5268. In some embodiments, the immunosuppressive gene is HLA-G and the microRNA is miR-133A, miR-148A, miR-148B, miR-152, miR-548q and / or miR-628-5p. One or more of them. See, eg, Schwich et al., 2019, Scientific Reports, 9:5407. In some embodiments, the present disclosure provides a method for treating cells that contain a disruption (e.g., complete or partial deletion) of a gene encoding a microRNA that binds to the 3'-UTR of an immunosuppressive gene and reduces its expression. plan. In some embodiments, the present disclosure provides miR-34a, miR-140, miR-200a, miR-200b / c, miR-142, miR-340, miR-383, miR-424(322), miR- 338-5p, miR-324-5p, miR-152, miR-200b, miR-138-5p, miR-195, miR-16, miR-15, miR-193a-3p, miR-497-5p, miR- 33a, miR17-5p, miR-155, miR-513, miR-133A, miR-148A, miR-148B, miR-152, miR-548q and / or miR-628-5p. Cells containing a disruption (eg, deletion) of the encoding gene are contemplated.

[0068] In some embodiments, disruption in the 3'-UTR of an allele encoding an immunosuppressive factor results in expression of the immunosuppressive factor in isolated cells, stem cells, and / or cells differentiated from isolated stem cells. (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least twice, at least 5x, at least 10x, at least 50x, at least 100x or more). In some embodiments, increased expression of an immunosuppressive factor is induced or increased by a cytokine, such as interferon gamma. In some embodiments, increased expression of the immunosuppressive factor is induced or increased by interferon gamma. In some embodiments, rather than having constitutive expression of an inserted transgene of the immunosuppressive factor in the cell, the immunosuppressive factor is responsive to a cytokine, such as interferon gamma, as contemplated herein. It may be advantageous to have. In some embodiments, any of the cells disclosed herein are exposed to a cytokine (eg, interferon gamma) upon transplantation into a subject. In certain embodiments, it is not necessary to expose any of the cells disclosed herein to a cytokine (eg, interferon gamma) prior to transplantation into a subject.

[0069] As used herein, "immunosuppressive factor" refers to a gene or molecule that attenuates the immune response. In some embodiments, the immunosuppressive factor inhibits the adaptive arm of the immune system. In some embodiments, the immunosuppressive factor inhibits the innate arm of the immune system. In some embodiments, the immunosuppressive factor attenuates normal immune responses during development. In some embodiments, immunosuppressive factors attenuate normal immune responses during disease states. In some embodiments, immunosuppressive factors are used to attenuate the immune response during tissue or cell transplantation. Non-limiting examples of immunosuppressive factors that can be used in accordance with the present disclosure include PDL1, PDL2, CD47, HLA-G, CTLA-4, HLA-C, HLA-E, C1 inhibitor, IL-10, IL- 35, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9 can be mentioned. In some embodiments, hypoimmune cells described herein are produced by disrupting the 3'UTR of one or more immunosuppressive factors.

[0070] In some embodiments, a cell described herein (eg, an isolated stem cell) comprises a disruption in the 3'-UTR of an allele encoding programmed death ligand 1 (PDL1). In some embodiments, the disruption is a homozygous modification. In some embodiments, the disruption is a heterozygous modification. "Programmed death ligand 1 (PDL1)" is an immunoinhibitory receptor ligand expressed by hematopoietic and non-hematopoietic cells such as T cells and B cells, as well as various types of tumor cells. The PDL1 protein is a type I transmembrane protein with an immunoglobulin V-like domain and a C-like domain. The interaction of this ligand with its receptor inhibits T cell activation and cytokine production. During inflammatory infections of normal tissues, this interaction is important for maintaining homeostasis of the immune response and preventing autoimmunity.

[0071] An example of the Homo sapiens PDL1 gene sequence is provided by NCBI gene ID: 29126 (SEQ ID NO: 28; corresponding to positions 5450542 to 5470554 of the Homo sapiens chromosome 9 sequence provided by NCBI accession number NC_000009.12). . Additionally, examples of human PDL1 transcript variants encoding different isoforms of the PDL1 protein include NCBI accession numbers NM_014143.4 (SEQ ID NO: 1), NM_001267706.2 (SEQ ID NO: 2), or NM_001314029.2 (SEQ ID NO: 3). is provided at.

[0072] Human PDL1 gene - NCBI gene ID: 29126 (SEQ ID NO: 28); 3’-UTR underline (SEQ ID NO: 87) GGGTGAGGGGCAGGAGAATGGGTATGGATGGAGGTAGAAGATGCAGTGTCATACAGTTTTTTTCTATCATGAAAATAACCACAGACTTACAGAAGAGAAAGAGCTAAAATGCCCGTCATTTTCAGTTGCATTTTAGTCTTGCATTAGTTGCAACCAGCTGGTTTCTGGGTACCCTAAGTAATAAAAATAGTTCCTCTGTAGAACTGTAGTATGTTTACCATAGAGTATTTTGCAAAATTTTTGGTAGAGGATGTTACATAATTTGCATGTGTTCATTTCTCCATTTACCTGTGGGAACAATTAAAATCCAGGAAAATGAGTATATTCAAATAATTTCCTCCCATTTAAGATGAGTCAGAGTAAATAATTCCTCCAATACTTAGAGAAGTATACCAAGAGATCCAGTGATGGTATAGAGTTGTCTGATGTTAAATAGGGAAGTAGAATATGGAAGGGGATTCCAATAGTCGTTGAAAAATTCCCCATAACCCCTTACATGGGGGAAAGTAGTGTTAACTGAGAGAGTAGAGATAAGCTGTTTCCAAAAATTATATTCTTAACAGGACTGAGATAGCCAGAATATAAGGATCAAGTTTCAATGACAGTAAGATCCTGAGATGGAGTTGATTTGCACAAAGAAATAATTGTTGCCAGCATGCATTTTGAATATTTCTCTGGAAAAAAAGATTAGTTGGCAGTAGAAATGGATAGAAATCAATAGATATTAAAATACCTCAGAATTTGGTTCATCTCTGGGAAAAGATGAAAAATAAAAGTGTATACTCCTCAAGAACATCTAGGATCAAAAGCATGTGCCCTACACTATTGAATTAATTAACCTCATAAGTTGGGACCTGTGGAATAAGGATGTCCACCAGACTTCCTAGGGATTACAAATGTTTCACAGAACTTGAAATTTAAACTTGGGTCACTGTATGGGATGTAGAGCTGTGCTATATGGAAATAAAAATGATTTCTTTTTCTCAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGGTAAGAATATCAGAAGGAATTGGGAAGTAAAAGTCAAAGGAAACAAAAAGCTAAAGCAATAACAAAGAGAAATCCATCAGTCATAATCTCCTCTCCTTTTAAAGAATGCTGGTTCCCCTTTGCCTCACAGCTAACACAAGAACTCCTCCACCGTCTGAGGAGGTTTAGGAGCAGGGAAGGGGAAGGAGTCAGCTTCATTTGCTAATCTTCTGTTGCCCTGCACCCTAGCAGCTCCTTGCAGCAGGGGACAAGGATGACTTAGGTGGATGGATAATTAATTGATTCTAAAATATTGTGTGTCAGTATTGTAATACTATGTTAATTGCACCATGCACGGTATCTCATTTAATCCCCCACCCCTTGCCATTACCAAAGAGAGAGAGAGAGAGAGAGAGAGAAATACTAGAATTTATCCTCATTTTACAGTAGAGAAAACAGAGGGTCAAGAAGATAATGTAAAGTGCCCAAGAACACACAGCTGATCACAAAAATCAAGCTTGGGGGCCATTAGCCTAACCACAGACCCTTACTCTTAACCCATCTGCTTCAATCCATTTTGCTACAAATGTTTACATTTATAAGCAGGGCAGAAAAACCTCATCCAGGTTATTGAACTAAGAAGAAAGTTATATTAAGGTTTCTAATTTTTTTAATGTAGTTAGAAACCAAACTTAACAATGAGCCCAAGTTTAAAGCAGTCTAATTAACCTGGACAAGCTCAGGCAAGTTTCATTCTGTGGCCCATAGCATCATCTGTGTTGTAAAGCTAAGTAGCAAATGTTGTTTGGGTCATGCTGGGGGACAAGCCATCCCAATTTGCTCAGGACTGAGGGGTTTTCCAGGATATCATGTAAGGATAATTGGGTACAAATATAACCTGCTGCTTTCTCTCATTTCAAATTTATCATTTATCATATCAGCAACTATGAGTTATGTTTTTTATTAGATTTCTTGTTACTTTTTCCCCAGACCACTTCCCATGAAATTAATATACTATTATCACTCTCCAGATACACATTTGGAGGAGACGTAA TCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCTCATCGACGCCTGTGACAGGGA GAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCATCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGCAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGTGAATG ATTTCTTTTGAAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGT AGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCCACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTATTCAAAAACCATTTATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATT GAATCTACAGATGTGAGCAAGACAAAGTACCTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAAACCCG AGCAGTGTTGCCAAGAGGAGGAAATAGGCCAATGTGGTCTGGGACGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTTACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACT GGAATTCCTTTTCTAGCATTATATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACTGCTACTGCCTTTCATTCATATGTTC TTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTTATTTATTTTAGTGTTTCTTATA ATTTAGACAACCACCATTTGTTAAGTATTTGCTTAGGACAGAGTTTGGATTTGTTTATGTTTGCTCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTA GCTACCCTGTGCCAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTACATGTG CATTTGTACAGTAATTGGTGTGACAGTGTTCTTTGTGTGAATTACAGGCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCTCCTTGTGGTTGGATTTGTAAGG ACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCA.

[0073] Human PDL1 transcript variant 1 - NM_014143.4 (SEQ ID NO: 1); 3’-UTR underline (SEQ ID NO: 78) AGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATT CAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACAT GTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCT GGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA TCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCTCATCGACGCCTGTGACAGGGA GAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCATCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGCAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGTGAATG ATTTCTTTTGAAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGT AGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCCACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTATTCAAAAACCATTTATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATT GAATCTACAGATGTGAGCAAGACAAAGTACCTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAAACCCG AGCAGTGTTGCCAAGAGGAGGAAATAGGCCAATGTGGTCTGGGACGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTTACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACT GGAATTCCTTTTCTAGCATTATATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACTGCTACTGCCTTTCATTCATATGTTC TTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTTATTTATTTTAGTGTTTCTTATA ATTTAGACAACCACCATTTGTTAAGTATTTGCTTAGGACAGAGTTTGGATTTGTTTATGTTTGCTCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTA GCTACCCTGTGCCAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTACATGTG CATTTGTACAGTAATTGGTGTGACAGTGTTCTTTGTGTGAATTACAGGCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCTCCTTGTGGTTGGATTTGTAAGG ACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCA.

[0074] Human PDL1 transcript variant 2 - NM_001267706.2 (SEQ ID NO: 2); 3’-UTR underline (SEQ ID NO: 79) AGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTA AGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAAT GATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA TCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCTCATCGACGCCTGTGACAGGGA GAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCATCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGCAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGTGAATG ATTTCTTTTGAAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGT AGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCCACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTATTCAAAAACCATTTATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATT GAATCTACAGATGTGAGCAAGACAAAGTACCTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAAACCCG AGCAGTGTTGCCAAGAGGAGGAAATAGGCCAATGTGGTCTGGGACGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTTACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACT GGAATTCCTTTTCTAGCATTATATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACTGCTACTGCCTTTCATTCATATGTTC TTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTTATTTATTTTAGTGTTTCTTATA ATTTAGACAACCACCATTTGTTAAGTATTTGCTTAGGACAGAGTTTGGATTTGTTTATGTTTGCTCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTA GCTACCCTGTGCCAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTACATGTG CATTTGTACAGTAATTGGTGTGACAGTGTTCTTTGTGTGAATTACAGGCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCTCCTTGTGGTTGGATTTGTAAGG ACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCA.

[0075] Human PDL1 transcript variant 4 - NM_001314029.2 (SEQ ID NO: 3); 3’-UTR underlined AGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATT ATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGC AGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGA CATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGGTAATATTCTGAATGTGTCCATTAAAATATGTCTAACACTGTCCCC TAGCACCTAG CATGATGTCTGCCTATCATAGTCATTCAGTGATTGTTGAATAAATGAATGAATGAATAACA.

[0076] In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises a deletion of the 3'-UTR. In some embodiments, the deletion comprises deletion of one or more fragments of the 3'-UTR. In some embodiments, the deletion is a complete deletion of the 3'-UTR. In some embodiments, the deletion is a partial deletion of the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises a sequence inversion in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises inversion of a fragment in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises an inversion of the entire 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises translocation of the entire 3'-UTR sequence. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 comprises one or more nucleotide substitutions in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 spans from any of the nucleotides corresponding to nucleotides 1000-1050 of SEQ ID NO: 1 to nucleotides corresponding to 3634 of SEQ ID NO: 1. ' Contains deletions that result in the loss of portions of the UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 spans from any of the nucleotides corresponding to nucleotides 1010-1050 of SEQ ID NO: 1 to nucleotides corresponding to 3634 of SEQ ID NO: 1. ' Contains deletions that result in the loss of portions of the UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL1 spans from any of the nucleotides corresponding to nucleotides 1010-1040 of SEQ ID NO: 1 to nucleotides corresponding to 3634 of SEQ ID NO: 1. ' Contains deletions that result in the loss of portions of the UTR. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 200, 300, 400 of the 3'UTR of the allele encoding PDL1 , 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 2200, 2300, 2400, 2500, or 2600 nucleotides (eg, contiguous nucleotides) are deleted. In some embodiments, SEQ ID NO: 1 and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700 from the portion corresponding to nucleotides 943 to 3634 of the sequence that are 100% identical; 2000, 2200, 2300, 2400, 2500, or 2600 nucleotides (eg, contiguous nucleotides) are deleted. In some embodiments, the present disclosure provides that the 3'-UTR of the PDL1 gene has a complete or partial deletion (e.g., at least 10 , 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 2200, 2300, 2400, 2500, or 2600 nucleotides, e.g. consecutive nucleotides ) to provide cells (e.g., stem cells) that have been disrupted. In some embodiments, the present disclosure provides that the 3'-UTR of the PDL1 gene has a complete or partial deletion (e.g., at least 10 , 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 2200, 2300, 2400, 2500, or 2600 nucleotides, e.g. consecutive nucleotides ) provides cells (stem cells) in which the 3'-UTR of the PDL1 gene has been destroyed. In some embodiments, the present disclosure uses a gene editing system that includes an RNA-guided endonuclease (e.g., Cas9 or Cas12) and an RNA guide that targets the sequences of SEQ ID NO: 13 and SEQ ID NO: 15 to A cell in which a portion of the cell's genome has been excised is contemplated. In some embodiments, the present disclosure provides gene editing systems that include an RNA-guided endonuclease (e.g., Cas9 or Cas12) and an RNA guide that targets the sequences of SEQ ID NO: 14 and SEQ ID NO: 15. A cell in which a portion of the cell's genome has been excised is contemplated.

[0077] In some embodiments, the present disclosure provides a method for treating cells in which SEQ ID NO: 31 (TCCAGCATTGGAACTTCTGATCT) or SEQ ID NO: 32 (TCTGATC) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. plan. In some embodiments, the sequence of SEQ ID NO: 31 or 32 is such that miR-140 is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 32 or 32 or an RNA equivalent or complementary sequence thereof. '-mutated in the UTR. In some embodiments, the present disclosure provides a method for treating cells in which SEQ ID NO: 33 (CCACCCTGTTGTGATAACCACTA) or SEQ ID NO: 34 (AACCACT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. plan. In some embodiments, the sequence of SEQ ID NO: 33 or 34 is such that miR-142 is unable to bind or has significantly reduced binding to SEQ ID NO: 33 or 34 or an RNA equivalent and / or complementary sequence thereof. It is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 35 (GCCACCCACTGTCCTTTTATAAT) or 36 (TTTATAA) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, the sequence of SEQ ID NO: 35 or 36 is such that miR-340 is unable to bind or has significantly reduced binding to SEQ ID NO: 35 or 36 or an RNA equivalent and / or its complementary sequence. It is mutated in the 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 37 (TTGGATTTGTAAGGCACTTTAT) or 38 (ACTTTAT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, the sequence of SEQ ID NO: 37 or 38 is such that miR-383 is unable to bind or has significantly reduced binding to SEQ ID NO: 37 or 38 or an RNA equivalent and / or complementary sequence thereof. , is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 39 (GGCTCATCGACGCCTGTGAC) or 40 (CCTGTGA) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, the sequence of SEQ ID NO: 39 or 40 is such that miR-513 is unable to bind or has significantly reduced binding to SEQ ID NO: 39 or 40 or an RNA equivalent and / or complementary sequence thereof. It is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure provides that one or more SEQ ID NO: 41 (AATAGCAGACCTCAGACTGCCA) or 42 (ACTGCCA) of the PDL1 3'-UTR is used to generate a sequence of SEQ ID NO: 43 (ACTCCCA), e.g. Cells containing nucleotide deletions, insertions, and / or substitutions are contemplated. In some embodiments, the sequence of SEQ ID NO: 41 or 42 is such that miR-34a is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 41 or 42 or an RNA equivalent and / or complementary sequence thereof. It is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure provides that SEQ ID NO: 44 (CAGTGTTGGAACGGGACAGTATTT), 45 (CAGTGTT), or 46 (CAGTATT) of the PDL1 3'-UTR is one or more nucleotide deletions, insertions, and / or Cells containing substitutions are contemplated. In some embodiments, miR-200a and / or miR-200b / c are unable to bind or have significantly reduced binding to SEQ ID NO: 44, 45 or 46, or RNA equivalents and / or complementary sequences thereof. As such, the sequence SEQ ID NO: 44, 45 or 46 is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 47 (CCAAACTAAACTTGCTGCTT) or 48 (TTGCTGCT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-424(322), miR-195, or miR497-5p is unable to bind, or does not significantly bind to SEQ ID NO: 47 or 48, or an RNA equivalent and / or its complement. The sequence SEQ ID NO: 47 or 48 is mutated in the PDL1 3'-UTR so that it is reduced. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 57 (TGTGAGCAAGACAAAGTAC) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the sequence of SEQ ID NO: 57 is in the PDL1 3'-UTR such that miR-33a is unable to bind or has significantly reduced binding to SEQ ID NO: 57 or an RNA equivalent and / or its complementary sequence. There is a mutation in In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 58 (GCATTAA) or 59 (AGCATTA) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. . In some embodiments, a sequence of SEQ ID NO: 58 and / or 59 such that miR155 is unable to bind or has significantly reduced binding to SEQ ID NO: 58 and / or 59 or an RNA equivalent and / or complementary sequence thereof. is mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 60 (ACTTAAAAGGCCCAAGCACTGAA) or 61 (GCACTG) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-152 is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 60 and / or 61, or an RNA equivalent and / or complementary sequence thereof. 61 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure provides a method for treating cells in which SEQ ID NO: 65, 62 (TATTTTGTTACTTGGTACACCAGCA) or 63 (ACACCAGC) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. plan. In some embodiments, miR-138-5p is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 62 and / or 63, or RNA equivalents and / or complementary sequences thereof. / or 63 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 64 (CGCCAAACTAAACTTGCTGCTT) or 65 (ACTTGCTGCT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-16, miR-15a, and / or miR15b are unable to bind or have significantly reduced binding to SEQ ID NO: 64 and / or 65, or RNA equivalents and / or complementary sequences thereof. As such, the sequences SEQ ID NO: 64 and / or 65 are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 66 (ATCCCTAATTTGAGGGTCAGTT) or 67 (TTTGAGGGTCAGT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-193a is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 66 and / or 67, or an RNA equivalent and / or complementary sequence thereof. 67 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 68 (GGTGTTGGATTTGTAAGGCACTTTA) or 69 (GCACTTT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-17-5p is unable to bind or has significantly reduced binding to SEQ ID NO: 68 and / or 69 or an RNA equivalent and / or its complementary sequence. 69 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 70 (AAGGAATGGGCCCGTGGGATGCA) or 71 (GGGATGC) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-324-5p is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 70 and / or 71, or RNA equivalents and / or complementary sequences thereof. or 71 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 72 (ATTTCTTTTGAAGATATATTGTA) or 73 (ATATTGT) of the PDL1 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. do. In some embodiments, miR-338-5p is unable to bind to, or has significantly reduced binding to, SEQ ID NO: 72 and / or 73, or RNA equivalents and / or complementary sequences thereof. / or 73 sequences are mutated in the PDL1 3'-UTR. In some embodiments, the present disclosure provides that the PDL1 3'-UTR SEQ ID NOs: 32, 34, 36, 38, 40, 42, 45, 48, 36, 58, 59, 61, 63, 65, 67, 69 , 71, and / or 73 in which 1, 2, 3, 4, 5, 6, or 7 or all nucleotides are deleted. In some embodiments, the present disclosure provides PDL1 3'-UTR SEQ ID NOs: 31, 33, 35, 37, 39, 41, 44, 47, 57, 60, 62, 64, 66, 68, 70, and / or any one of 72 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24, or all nucleotides are deleted.For example, Xie et al., 2017 PLOS One, DOI:10.1371 / journal.pone.0168822; Zhao et al., 2016, Oncotarget, 7(29):45370~84; He et al., 2018 Biomedicine and Pharmacology, 98:95 ~101 pages; Tao et al. 2018, Cell Physiol Biochem., 48:801~814; Kao et al., 2017, J. Thoracic Oncology, 12(9):1421~1433; Audrito et al., 2017, Oncotarget, 8(9):15894 ~15911 pages; Holla et al., 2016, Scientific Reports, 6(24193);Danbaran et al., 2020, International Immunopharmacology, 84:106594;Gong et al., 2009, J Immunol., 182(3):1325-1333;Chen et al., 2014, Nat. Commun. , 5:5241; Wang et al., 2015, Cellular Signaling, 27(3):443~452; is incorporated herein by reference in its entirety. Because the sequences of SEQ ID NOs: 1, 2, 31, 32-42, 44-45, 47-48, and / or 57-73 of PDL1 3'-UTR are derived from nucleotide sequences naturally present in cells, It is noted that the nucleic acids within a cell may have some differences (eg, polymorphisms) compared to these reference sequences. Accordingly, the present disclosure provides that cells have a reference sequence of any of SEQ ID NOs: 1, 2, 31, 32-42, 44-45, 47-48, and / or 57-73 of PDL1 3'-UTR before modification. It is contemplated that nucleotides may have a difference of 1, 2, 3, 4, 5, or 6 or fewer nucleotides as compared to . In some embodiments, the cell is heterozygous for any one or combination of the above genetic elements listed in this paragraph. In some embodiments, the cell is homozygous for any one or combination of the above genetic elements listed in this paragraph.

[0078] In some embodiments, disruption in the 3'-UTR of an allele encoding PDL1 results in increased expression of PDL1 (e.g., at least 10%, at least 20%, at least 30%, less At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times or more increase). In some embodiments, increased expression of PDL1 is induced or increased by interferon gamma.

[0079] In some embodiments, a cell described herein (eg, an isolated stem cell) comprises a disruption in the 3'-UTR of an allele encoding surface antigen classification 47 (CD47). In some embodiments, the disruption is a homozygous modification. In some embodiments, the disruption is a heterozygous modification. “Surface antigen classification 47 (CD47)” belongs to the immunoglobulin superfamily, partners with membrane integrins, and also binds to the ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). CD47 acts as a "don't eat me" signal to macrophages in the immune system. Increased expression of CD47 on cells administered to a subject as a cell-based therapy protects the cells from phagocytosis by macrophages.

[0080] An example of the Homo sapiens CD47 gene sequence is NCBI gene ID: 961 (SEQ ID NO: 29; corresponds to the sequence complementary to positions 108043091 to 108094200 of the Homo sapiens chromosome 3 sequence provided by NCBI accession number NC_000003.12) provided to. Additionally, examples of human CD47 transcript variants encoding different isoforms of the CD47 protein include NCBI accession numbers NM_001777.4 (SEQ ID NO: 4), NM_198793.3 (SEQ ID NO: 5), or NM_001382306.1 (SEQ ID NO: 6). provided to.

[0081] Human CD47 gene - NCBI gene ID: 961 (SEQ ID NO: 29); 3’-UTR underline (SEQ ID NO: 88) CCAGGAATTGTGGCATGGTTTGTTCATGTTTACATTCTGATGTCCTATTTTTTTTTTTTAATTTCTATGTCCTTTCCTTTTCCTTGGTGGTGTCATTGTTCTGTAGCTGTATGAAGAAACTAAACTTTTCTCCATTTTCAGGAAAGCAATCTAAGAATCTTGAGTGCCTCTTCCTTTGTTAATTTCTCTTAAGATGTGACTTTTTTAAACTACTGCATCAGGAAATATTGTAAAACAGTTTTGCCTTGAATATTTGTGATGAAATCTACGATGATCTTCAAGATTCTCTTAATTTTGCTAATATTCAGCTGATCAGAATTTGTTTTTAAAATGTCTGGCTGGTGGGTACTTCCCACTGACAACTGCTTATTGCTTACAGTATGTCTGCCTTGTCAATGAATGAGGTTCAGGGTGCTTCCTAGGGATCAGAGTCAGTACCATTTTTCTCTTTCATCTACAGCTGATCAGATGTTTATTTTACTTACATTAAATGAATGATGGAGATCCAAAGTGAATATTATAGAATATTATTCTAGGATCAACATCTTTTGCTTTGAAAAATCAACATCTCTTGGCTTTTCCTCAGCCAACCCAGCAAACAGAGATTATCAGACTCTGTTGATTTTTTACTTTCATTTGGCATTGGCCTTTTTCTTACTGAAATTAAAAAGGCTAATGATTTGCCTGGTTTCTGTCTCTGACCTTTGCAGGTCTATTTTCTTAATTTTTAGATACTATATATCTGAAACTTTTTTTAATGTGTCAACTTTTTAATGGATAGAAAATAGACACGAATAGTGATTATGTGTTCATTTTTCAATTTTCCAGAATAACTGA AGTGAAGTGATGGACTCCGATTTGGAGAGTAGTAAGACGTGAAAGGAATACACTTGTGTTTAAGCACCATGGCCTTGATGATTCACTGTTGGGGAGAAGAAACAAGAAAAGTAACTGGTTGTCACCTATGAGACCCTTACGTGATTGTTAGTTAAGTTTTTATTCAAAGCAGCTGTAATTTAGTTAATAAAATAATTATGATCTATGTTGTTTGCCCAATTGAGATCCAGTTTTTTGTTGTTATTTTTAATCAATTAGGGGCAATAGTAGAATGGACAATTTCCAAGAATGATGCCTTTCAGGTCCTAGGGCCTCTGGCCTCTAGGTAACCAGTTTAAATTGGTTCAGGGTGATAACTACTTAGCACTGCCCTGGTGATTACCCAGAGATATCTATGAAAACCAGTGGCTTCCATCAAACCTTTGCCAACTCAGGTTCACAGCAGCTTTGGGCAGTTATGGCAGTATGGCATTAGCTGAGAGGTGTCTGCCACTTCTGGGTCAATGGAATAATAAATTAAGTACAGGCAGGAATTTGGTTGGGAGCATCTTGTATGATCTCCGTATGATGTGATATTGATGGAGATAGTGGTCCTCATTCTTGGGGGTTGCCATTCCCACATTCCCCCTTCAACAAACAGTGTAACAGGTCCTTCCCAGATTTAGGGTACTTTTATTGATGGATATGTTTTCCTTTTATTCACATAACCCCTTGAAACCCTGTCTTGTCCTCCTGTTACTTGCTTCTGCTGTACAAGATGTAGCACCTTTTCTCCTCTTTGAACATGGTCTAGTGACACGGTAGCACCAGTTGCAGGAAGGAGCCAGACTTGTTCTCAGAGCACTGTGTTCACACTTTTCAGCAAAAATAGCTATGGTTGTAACATATGTATTCCCTTCCTCTGATTTGAAGGCAAAAATCTACAGTGTTTCTTCACTTCTTTTCTGATCTGGGGCATGAAAAAAGCAAGATTGAAATTTGAACTATGAGTCTCCTGCATGGCAACAAAATGTGTGTCACCATCAGGCCAACAGGCCAGCCCTTGAATGGGGATTTATTACTGTTGTATCTATGTTGCATGATAAACATTCATCACCTTCCTCCTGTAGTCCTGCCTCGTACTCCCCTTCCCCTATGATTGAAAAGTAAACAAAACCCACATTTCCTATCCTGGTTAGAAGAAAATTAATGTTCTGACAGTTGTGATCGCCTGGAGTACTTTTAGACTTTTAGCATTCGTTTTTTACCTGTTTGTGGATGTGTGTTTGTATGTGCATACGTATGAGATAGGCACATGCATCTTCTGTATGGACAAAGGTGGGGTACCTACAGGAGAGCAAAGGTTAATTTTGTGCTTTTAGTAAAAACATTTAAATACAAAGTTCTTTATTGGGTGGAATTATATTTGATGCAAATATTTGATCACTTAAAACTTTTAAAACTTCTAGGTAATTTGCCACGCTTTTTGACTGCTCACCAATACCCTGTAAAAATACGTAATTCTTCCTGTTTGTGTAATAAGATATTCATATTTGTAGTTGCATTAATAATAGTTATTTCTTAGTCCATCAGATGTTCCCGTGTGCCTCTTTTATGCCAAATTGATTGTCATATTTCATGTTGGGACCAAGTAGTTTGCCCATGGCAAACCTAAATTTATGACCTGCTGAGGCCTCTCAGAAAACTGAGCATACTAGCAAGACAGCTCTTCTTGAAAAAAAAAATATGTATACACAAATATATACGTATATCTATATATACGTATGTATATACACACATGTATATTCTTCCTTGATTGTGTAGCTGTCCAAAATAATAACATATATAGAGGGAGCTGTATTCCTTTATACAAATCTGATGGCTCCTGCAGCACTTTTTCCTTCTGAAAATATTTACATTTTGCTAACCTAGTTTGTTACTTTAAAAATCAGTTTTGATGAAAGGAGGGAAAAGCAGATGGACTTGAAAAAGATCCAAGCTCCTATTAGAAAAGGTATGAAAATCTTTATAGTAAAATTTTTTATAAACTAAAGTTGTACCTTTTAATATGTAGTAAACTCTCATTTATTTGGGGTTCGCTCTTGGATCTCATCCATCCATTGTGTTCTCTTTAATGCTGCCTGCCTTTTGAGGCATTCACTGCCCTAGACAATGCCACCAGAGATAGTGGGGGAAATGCCAGATGAAACCAACTCTTGCTCTCACTAGTTGTCAGCTTCTCTGGATAAGTGACCACAGAAGCAGGAGTCCTCCTGCTTGGGCATCATTGGGCCAGTTCCTTCTCTTTAAATCAGATTTGTAATGGCTCCCAAATTCCATCACATCACATTTAAATTGCAGACAGTGTTTTGCACATCATGTATCTGTTTTGTCCCATAATATGCTTTTTACTCCCTGATCCCAGTTTCTGCTGTTGACTCTTCCATTCAGTTTTATTTATTGTGTGTTCTCACAGTGACACCATTTGTCCTTTTCTGCAACAACCTTTCCAGCTACTTTTGCCAAATTCTATTTGTCTTCTCCTTCAAAACATTCTCCTTTGCAGTTCCTCTTCATCTGTGTAGCTGCTCTTTTGTCTCTTAACTTACCATTCCTATAGTACTTTATGCATCTCTGCTTAGTTCTATTAGTTTTTTGGCCTTGCTCTTCTCCTTGATTTTAAAATTCCTTCTATAGCTAGAGCTTTTCTTTCTTTCATTCTCTCTTCCTGCAGTGTTTTGCATACATCAGAAGCTAGGTACATAAGTTAAATGATTGAGAGTTGGCTGTATTTAGATTTATCACTTTTTAATAGGGTGAGCTTGAGAGTTTTCTTTCTTTCTGTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTAATTTCACATGCTCTAAAAACCTTCAAAGGTGATTATTTTTCTCCTGGAAACTCCAGGTCCATTCTGTTTAAATCCCTAAGAATGTCAGAATTAAAATAACAGGGCTATCCCGTAATTGGAAATATTTCTTTTTTCAGGATGCTATAGTCAATTTAGTAAGTGACCACCAAATTGTTATTTGCACTAACAAAGCTCAAAACACGATAAGTTTACTCCTCCATCTCAGTAATAAAAATTAAGCTGTAATCAACCTTCTAGGTTTCTCTTGTCTTAAAATGGGTATTCAAAAATGGGGATCTGTGGTGTATGTATGGAAACACATACTCCTTAATTTACCTGTTGTTGGAAACTGGAGAAATGATTGTCGGGCAACCGTTTATTTTTTATTGTATTTTATTTGGTTGAGGGATTTTTTTATAAACAGTTTTACTTGTGTCATATTTTAAAATTACTAACTGCCATCACCTGCTGGGGTCCTTTGTTAGGTCATTTTCAGTGACTAATAGGGATAATCCAGGTAACTTTGAAGAGATGAGCAGTGAGTGACCAGGCAGTTTTTCTGCCTTTAGCTTTGACAGTTCTTAATTAAGATCATTGAAGACCAGCTTTCTCATAAATTTCTCTTTTTGAAAAAAAGAAAGCATTTGTACTAAGCTCCTCTGTAAGACAACATCTTAAATCTTAAAAGTGTTGTTATCATGACTGGTGAGAGAAGAAAACATTTTGTTTTTATTAAATGGAGCATTATTTACAAAAAGCCATTGTTGAGAATTAGATCCCACATCGTATAAATATCTATTAACCATTCTAAATAAAGAGAACTCCAGTGTTGCTATGTGCAAGATCCTCTCTTGGAGCTTTTTTGCATAGCAATTAAAGGTGTGCTATTTGTCAGTAGCCATTTTTTTGCAGTGATTTGAAGACCAAAGTTGTTTTACAGCTGTGTTACCGTTAAAGGTTTTTTTTTTTATATGTATTAAATCAATTTATCACTGTTTAAAGCTTTGAATATCTGCAATCTTTGCCAAGGTACTTTTTTATTTAAAAAAAAACATAACTTTGTAAATATTACCCTGTAATATTATATATACTTAATAAAACATTTTAAGCTATTTTGTTGGGCTATTTCTATTGCTGCTACAGCAGACCACAAGCACATTTCTGAAAAATTTAATTTATTAATGTATTTTTAAGTTGCTTATATTCTAGGTAACAATGTAAAGAATGATTTAAAATATTAATTATGAATTTTTTGAGTATAATACCCAATAAGCTTTTAATTAGAGCAGAGTTTTAATTAAAAGTTTTAAATCAGTCCAA。

[0082] Human CD47 transcript variant 1 - NM_001777.4 (SEQ ID NO: 4); 3’-UTR underlined (SEQ ID NO: 80) GCAGCCTGGGCAGTGGGTCCTGCCTGTGACGCGCGGCGGCGGTCGGTCCTGCCTGTAACGGCGGCGGCGGCTGCTGCTCCGGACACCTGCGGCGGCGGCGGCGACCCCGCGGCGGGCGCGGAGATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGCTCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTGTCGTCATTCCATGCTTTGTT ACTAATATGGAGGCACAAAACACTACTGAAGTATACGTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCTTTGATGGAGCTCTAAACAAGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTGAAGTCTCACAATTACTAAAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGAAACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGCTAAAATATC GTGTTGTTTCATGGTTTTCTCCAAATGAAAATATTCTTATTGTTATTTTCCAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTAAAACACTTAAATATAGATCCGGTGGTATGGATGAGAAAACAATTGCTTTACTTGTTGCTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCAGGTGAATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATA TTAATATTACTTCACTACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATTGCCATATTGGTTATTCAGGTGATAGCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTGAGTATCTTAGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGCTTCCAATCAGAAGACTATACAACCTCCTAGGAAAGCTGTAGA GGAACCCCTTAATGCATTCAAAGAATCAAAAGGAATGATGAATGATGAATAA CTGAAGTGAAGTGATGGACTCCGATTTGGAGAGTAGTAAGACGTGAAAGGAATACACTTGTGTTTAAGCACCATGGCCTTGATGATTCACTGTTGGGGAGAAGAAACAAGAAAAGTAACTGGTTGTCACCTATGAGACCCTTACGTGATTGTTAGTTAAGTTTTTATTCAAAGCAGCTGTAATTTAGTTAATAAAATAATTATGATCTATGTTGTTTGCCCAATTGAGATCCAGTTTTTTGTTGTTATTTTTAATCAATTAGGGGCAATAGTAGAATGGACAATTTCCAAGAATGATGCCTTTCAGGTCCTAGGGCCTCTGGCCTCTAGGTAACCAGTTTAAATTGGTTCAGGGTGATAACTACTTAGCACTGCCCTGGTGATTACCCAGAGATATCTATGAAAACCAGTGGCTTCCATCAAACCTTTGCCAACTCAGGTTCACAGCAGCTTTGGGCAGTTATGGCAGTATGGCATTAGCTGAGAGGTGTCTGCCACTTCTGGGTCAATGGAATAATAAATTAAGTACAGGCAGGAATTTGGTTGGGAGCATCTTGTATGATCTCCGTATGATGTGATATTGATGGAGATAGTGGTCCTCATTCTTGGGGGTTGCCATTCCCACATTCCCCCTTCAACAAACAGTGTAACAGGTCCTTCCCAGATTTAGGGTACTTTTATTGATGGATATGTTTTCCTTTTATTCACATAACCCCTTGAAACCCTGTCTTGTCCTCCTGTTACTTGCTTCTGCTGTACAAGATGTAGCACCTTTTCTCCTCTTTGAACATGGTCTAGTGACACGGTAGCACCAGTTGCAGGAAGGAGCCAGACTTGTTCTCAGAGCACTGTGTTCACACTTTTCAGCAAAAATAGCTATGGTTGTAACATATGTATTCCCTTCCTCTGATTTGAAGGCAAAAATCTACAGTGTTTCTTCACTTCTTTTCTGATCTGGGGCATGAAAAAAGCAAGATTGAAATTTGAACTATGAGTCTCCTGCATGGCAACAAAATGTGTGTCACCATCAGGCCAACAGGCCAGCCCTTGAATGGGGATTTATTACTGTTGTATCTATGTTGCATGATAAACATTCATCACCTTCCTCCTGTAGTCCTGCCTCGTACTCCCCTTCCCCTATGATTGAAAAGTAAACAAAACCCACATTTCCTATCCTGGTTAGAAGAAAATTAATGTTCTGACAGTTGTGATCGCCTGGAGTACTTTTAGACTTTTAGCATTCGTTTTTTACCTGTTTGTGGATGTGTGTTTGTATGTGCATACGTATGAGATAGGCACATGCATCTTCTGTATGGACAAAGGTGGGGTACCTACAGGAGAGCAAAGGTTAATTTTGTGCTTTTAGTAAAAACATTTAAATACAAAGTTCTTTATTGGGTGGAATTATATTTGATGCAAATATTTGATCACTTAAAACTTTTAAAACTTCTAGGTAATTTGCCACGCTTTTTGACTGCTCACCAATACCCTGTAAAAATACGTAATTCTTCCTGTTTGTGTAATAAGATATTCATATTTGTAGTTGCATTAATAATAGTTATTTCTTAGTCCATCAGATGTTCCCGTGTGCCTCTTTTATGCCAAATTGATTGTCATATTTCATGTTGGGACCAAGTAGTTTGCCCATGGCAAACCTAAATTTATGACCTGCTGAGGCCTCTCAGAAAACTGAGCATACTAGCAAGACAGCTCTTCTTGAAAAAAAAAATATGTATACACAAATATATACGTATATCTATATATACGTATGTATATACACACATGTATATTCTTCCTTGATTGTGTAGCTGTCCAAAATAATAACATATATAGAGGGAGCTGTATTCCTTTATACAAATCTGATGGCTCCTGCAGCACTTTTTCCTTCTGAAAATATTTACATTTTGCTAACCTAGTTTGTTACTTTAAAAATCAGTTTTGATGAAAGGAGGGAAAAGCAGATGGACTTGAAAAAGATCCAAGCTCCTATTAGAAAAGGTATGAAAATCTTTATAGTAAAATTTTTTATAAACTAAAGTTGTACCTTTTAATATGTAGTAAACTCTCATTTATTTGGGGTTCGCTCTTGGATCTCATCCATCCATTGTGTTCTCTTTAATGCTGCCTGCCTTTTGAGGCATTCACTGCCCTAGACAATGCCACCAGAGATAGTGGGGGAAATGCCAGATGAAACCAACTCTTGCTCTCACTAGTTGTCAGCTTCTCTGGATAAGTGACCACAGAAGCAGGAGTCCTCCTGCTTGGGCATCATTGGGCCAGTTCCTTCTCTTTAAATCAGATTTGTAATGGCTCCCAAATTCCATCACATCACATTTAAATTGCAGACAGTGTTTTGCACATCATGTATCTGTTTTGTCCCATAATATGCTTTTTACTCCCTGATCCCAGTTTCTGCTGTTGACTCTTCCATTCAGTTTTATTTATTGTGTGTTCTCACAGTGACACCATTTGTCCTTTTCTGCAACAACCTTTCCAGCTACTTTTGCCAAATTCTATTTGTCTTCTCCTTCAAAACATTCTCCTTTGCAGTTCCTCTTCATCTGTGTAGCTGCTCTTTTGTCTCTTAACTTACCATTCCTATAGTACTTTATGCATCTCTGCTTAGTTCTATTAGTTTTTTGGCCTTGCTCTTCTCCTTGATTTTAAAATTCCTTCTATAGCTAGAGCTTTTCTTTCTTTCATTCTCTCTTCCTGCAGTGTTTTGCATACATCAGAAGCTAGGTACATAAGTTAAATGATTGAGAGTTGGCTGTATTTAGATTTATCACTTTTTAATAGGGTGAGCTTGAGAGTTTTCTTTCTTTCTGTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTAATTTCACATGCTCTAAAAACCTTCAAAGGTGATTATTTTTCTCCTGGAAACTCCAGGTCCATTCTGTTTAAATCCCTAAGAATGTCAGAATTAAAATAACAGGGCTATCCCGTAATTGGAAATATTTCTTTTTTCAGGATGCTATAGTCAATTTAGTAAGTGACCACCAAATTGTTATTTGCACTAACAAAGCTCAAAACACGATAAGTTTACTCCTCCATCTCAGTAATAAAAATTAAGCTGTAATCAACCTTCTAGGTTTCTCTTGTCTTAAAATGGGTATTCAAAAATGGGGATCTGTGGTGTATGTATGGAAACACATACTCCTTAATTTACCTGTTGTTGGAAACTGGAGAAATGATTGTCGGGCAACCGTTTATTTTTTATTGTATTTTATTTGGTTGAGGGATTTTTTTATAAACAGTTTTACTTGTGTCATATTTTAAAATTACTAACTGCCATCACCTGCTGGGGTCCTTTGTTAGGTCATTTTCAGTGACTAATAGGGATAATCCAGGTAACTTTGAAGAGATGAGCAGTGAGTGACCAGGCAGTTTTTCTGCCTTTAGCTTTGACAGTTCTTAATTAAGATCATTGAAGACCAGCTTTCTCATAAATTTCTCTTTTTGAAAAAAAGAAAGCATTTGTACTAAGCTCCTCTGTAAGACAACATCTTAAATCTTAAAAGTGTTGTTATCATGACTGGTGAGAGAAGAAAACATTTTGTTTTTATTAAATGGAGCATTATTTACAAAAAGCCATTGTTGAGAATTAGATCCCACATCGTATAAATATCTATTAACCATTCTAAATAAAGAGAACTCCAGTGTTGCTATGTGCAAGATCCTCTCTTGGAGCTTTTTTGCATAGCAATTAAAGGTGTGCTATTTGTCAGTAGCCATTTTTTTGCAGTGATTTGAAGACCAAAGTTGTTTTACAGCTGTGTTACCGTTAAAGGTTTTTTTTTTTATATGTATTAAATCAATTTATCACTGTTTAAAGCTTTGAATATCTGCAATCTTTGCCAAGGTACTTTTTTATTTAAAAAAAAACATAACTTTGTAAATATTACCCTGTAATATTATATATACTTAATAAAACATTTTAA GCTATTTTGTTGGGCTATTTCTATTGCTGCTACAGCAGACCACAAGCACATTTCTGAAAAATTTAATTTATTAATGTATTTTTAAGTTGCTTATATTCTAGGTAACAATGTAAAGAATGATTTAAAATATTAATTATGAATTTTTTGAGTATAATACCAATAAGCTTTTAATTAGAGCAGAGTTTTAATTAAAAGTTTTAAATCAGTCCAA.

[0083] Human CD47 transcript variant 2 - NM_198793.3 (SEQ ID NO: 5); 3’-UTR underlined (SEQ ID NO: 81) GCAGCCTGGGCAGTGGGTCCTGCCTGTGACGCGCGGCGGCGGTCGGTCCTGCCTGTAACGGCGGCGGCGGCTGCTGCTCCGGACACCTGCGGCGGCGGCGGCGACCCCGCGGCGGGCGCGGAGATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGCTCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTGTCGTCATTCCATGCTTTGTTACT AATATGGAGGCACAAAACACTACTGAAGTATACGTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCTTTGATGGAGCTCTAAACAAGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTGAAGTCTCACAATTACTAAAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGAAACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGCTAAAATATCGTG TTGTTTCATGGTTTTCTCCAAATGAAAATATTCTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTAAAACACTTAAATATAGATCCGGTGGTATGGATGAGAAAACAATTGCTTTACTTGTTGCTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCAGGTGAATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATA TTAATATTACTTCACTACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATTGCCATATTGGTTATTCAGGTGATAGCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTGAGTATCTTAGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGCTTCCAATCAGAAGACTATACAACCTCCTAGGAATAACTGA AGTGAAGTGATGGACTCCGATTTGGAGAGTAGTAAGACGTGAAAGGAATACACTTGTGTTTAAGCACCATGGCCTTGATGATTCACTGTTGGGGAGAAGAAACAAGAAAAGTAACTGGTTGTCACCTATGAGACCCTTACGTGATTGTTAGTTAAGTTTTTATTCAAAGCAGCTGTAATTTAGTTAATAAAATAATTATGATCTATGTTGTTTGCCCAATTGAGATCCAGTTTTTTGTTGTTATTTTTAATCAATTAGGGGCAATAGTAGAATGGACAATTTCCAAGAATGATGCCTTTCAGGTCCTAGGGCCTCTGGCCTCTAGGTAACCAGTTTAAATTGGTTCAGGGTGATAACTACTTAGCACTGCCCTGGTGATTACCCAGAGATATCTATGAAAACCAGTGGCTTCCATCAAACCTTTGCCAACTCAGGTTCACAGCAGCTTTGGGCAGTTATGGCAGTATGGCATTAGCTGAGAGGTGTCTGCCACTTCTGGGTCAATGGAATAATAAATTAAGTACAGGCAGGAATTTGGTTGGGAGCATCTTGTATGATCTCCGTATGATGTGATATTGATGGAGATAGTGGTCCTCATTCTTGGGGGTTGCCATTCCCACATTCCCCCTTCAACAAACAGTGTAACAGGTCCTTCCCAGATTTAGGGTACTTTTATTGATGGATATGTTTTCCTTTTATTCACATAACCCCTTGAAACCCTGTCTTGTCCTCCTGTTACTTGCTTCTGCTGTACAAGATGTAGCACCTTTTCTCCTCTTTGAACATGGTCTAGTGACACGGTAGCACCAGTTGCAGGAAGGAGCCAGACTTGTTCTCAGAGCACTGTGTTCACACTTTTCAGCAAAAATAGCTATGGTTGTAACATATGTATTCCCTTCCTCTGATTTGAAGGCAAAAATCTACAGTGTTTCTTCACTTCTTTTCTGATCTGGGGCATGAAAAAAGCAAGATTGAAATTTGAACTATGAGTCTCCTGCATGGCAACAAAATGTGTGTCACCATCAGGCCAACAGGCCAGCCCTTGAATGGGGATTTATTACTGTTGTATCTATGTTGCATGATAAACATTCATCACCTTCCTCCTGTAGTCCTGCCTCGTACTCCCCTTCCCCTATGATTGAAAAGTAAACAAAACCCACATTTCCTATCCTGGTTAGAAGAAAATTAATGTTCTGACAGTTGTGATCGCCTGGAGTACTTTTAGACTTTTAGCATTCGTTTTTTACCTGTTTGTGGATGTGTGTTTGTATGTGCATACGTATGAGATAGGCACATGCATCTTCTGTATGGACAAAGGTGGGGTACCTACAGGAGAGCAAAGGTTAATTTTGTGCTTTTAGTAAAAACATTTAAATACAAAGTTCTTTATTGGGTGGAATTATATTTGATGCAAATATTTGATCACTTAAAACTTTTAAAACTTCTAGGTAATTTGCCACGCTTTTTGACTGCTCACCAATACCCTGTAAAAATACGTAATTCTTCCTGTTTGTGTAATAAGATATTCATATTTGTAGTTGCATTAATAATAGTTATTTCTTAGTCCATCAGATGTTCCCGTGTGCCTCTTTTATGCCAAATTGATTGTCATATTTCATGTTGGGACCAAGTAGTTTGCCCATGGCAAACCTAAATTTATGACCTGCTGAGGCCTCTCAGAAAACTGAGCATACTAGCAAGACAGCTCTTCTTGAAAAAAAAAATATGTATACACAAATATATACGTATATCTATATATACGTATGTATATACACACATGTATATTCTTCCTTGATTGTGTAGCTGTCCAAAATAATAACATATATAGAGGGAGCTGTATTCCTTTATACAAATCTGATGGCTCCTGCAGCACTTTTTCCTTCTGAAAATATTTACATTTTGCTAACCTAGTTTGTTACTTTAAAAATCAGTTTTGATGAAAGGAGGGAAAAGCAGATGGACTTGAAAAAGATCCAAGCTCCTATTAGAAAAGGTATGAAAATCTTTATAGTAAAATTTTTTATAAACTAAAGTTGTACCTTTTAATATGTAGTAAACTCTCATTTATTTGGGGTTCGCTCTTGGATCTCATCCATCCATTGTGTTCTCTTTAATGCTGCCTGCCTTTTGAGGCATTCACTGCCCTAGACAATGCCACCAGAGATAGTGGGGGAAATGCCAGATGAAACCAACTCTTGCTCTCACTAGTTGTCAGCTTCTCTGGATAAGTGACCACAGAAGCAGGAGTCCTCCTGCTTGGGCATCATTGGGCCAGTTCCTTCTCTTTAAATCAGATTTGTAATGGCTCCCAAATTCCATCACATCACATTTAAATTGCAGACAGTGTTTTGCACATCATGTATCTGTTTTGTCCCATAATATGCTTTTTACTCCCTGATCCCAGTTTCTGCTGTTGACTCTTCCATTCAGTTTTATTTATTGTGTGTTCTCACAGTGACACCATTTGTCCTTTTCTGCAACAACCTTTCCAGCTACTTTTGCCAAATTCTATTTGTCTTCTCCTTCAAAACATTCTCCTTTGCAGTTCCTCTTCATCTGTGTAGCTGCTCTTTTGTCTCTTAACTTACCATTCCTATAGTACTTTATGCATCTCTGCTTAGTTCTATTAGTTTTTTGGCCTTGCTCTTCTCCTTGATTTTAAAATTCCTTCTATAGCTAGAGCTTTTCTTTCTTTCATTCTCTCTTCCTGCAGTGTTTTGCATACATCAGAAGCTAGGTACATAAGTTAAATGATTGAGAGTTGGCTGTATTTAGATTTATCACTTTTTAATAGGGTGAGCTTGAGAGTTTTCTTTCTTTCTGTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTAATTTCACATGCTCTAAAAACCTTCAAAGGTGATTATTTTTCTCCTGGAAACTCCAGGTCCATTCTGTTTAAATCCCTAAGAATGTCAGAATTAAAATAACAGGGCTATCCCGTAATTGGAAATATTTCTTTTTTCAGGATGCTATAGTCAATTTAGTAAGTGACCACCAAATTGTTATTTGCACTAACAAAGCTCAAAACACGATAAGTTTACTCCTCCATCTCAGTAATAAAAATTAAGCTGTAATCAACCTTCTAGGTTTCTCTTGTCTTAAAATGGGTATTCAAAAATGGGGATCTGTGGTGTATGTATGGAAACACATACTCCTTAATTTACCTGTTGTTGGAAACTGGAGAAATGATTGTCGGGCAACCGTTTATTTTTTATTGTATTTTATTTGGTTGAGGGATTTTTTTATAAACAGTTTTACTTGTGTCATATTTTAAAATTACTAACTGCCATCACCTGCTGGGGTCCTTTGTTAGGTCATTTTCAGTGACTAATAGGGATAATCCAGGTAACTTTGAAGAGATGAGCAGTGAGTGACCAGGCAGTTTTTCTGCCTTTAGCTTTGACAGTTCTTAATTAAGATCATTGAAGACCAGCTTTCTCATAAATTTCTCTTTTTGAAAAAAAGAAAGCATTTGTACTAAGCTCCTCTGTAAGACAACATCTTAAATCTTAAAAGTGTTGTTATCATGACTGGTGAGAGAAGAAAACATTTTGTTTTTATTAAATGGAGCATTATTTACAAAAAGCCATTGTTGAGAATTAGATCCCACATCGTATAAATATCTATTAACCATTCTAAATAAAGAGAACTCCAGTGTTGCTATGTGCAAGATCCTCTCTTGGAGCTTTTTTGCATAGCAATTAAAGGTGTGCTATTTGTCAGTAGCCATTTTTTTGCAGTGATTTGAAGACCAAAGTTGTTTTACAGCTGTGTTACCGTTAAAGGTTTTTTTTTTTATATGTATTAAATCAATTTATCACTGTTTAAAGCTTTGAATATCTGCAATCTTTGCCAAGGTACTTTTTTATTTAAAAAAAAACATAACTTTGTAAATATTACCCTGTAATATTATATATACTTAATAAAACATTTTAAGCTATTTTGTTGGGCTATTTCTATTGCTGCTACAGCAGACCACAAGCACATTTCTGAA AAATTTAATTTATTAATGTATTTTTAAGTTGCTTATATTCTAGGTAACAATGTAAAGAATGATTTAAAATATTAATTATGAATTTTTTGAGTATAATACCAATAAGCTTTTAATTAGAGCAGAGTTTTAATTAAAAGTTTTAAATCAGTCCAA.

[0084] Human CD47 transcript variant 3 - NM_001382306.1 (SEQ ID NO: 6); 3’-UTR underline (SEQ ID NO: 82) GCAGCCTGGGCAGTGGGTCCTGCCTGTGACGCGCGGCGGCGGTCGGTCCTGCCTGTAACGGCGGCGGCGGCTGCTGCTCCGGACACCTGCGGCGGCGGCGGCGACCCCGCGGCGGGCGCGGAGATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGCTCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTGTCGTCATTCCATGCTTTGTTACT AATATGGAGGCACAAAACACTACTGAAGTATACGTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCTTTGATGGAGCTCTAAACAAGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTGAAGTCTCACAATTACTAAAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGAAACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGCTAAAATATCGTG TTGTTTCATGGTTTTCTCCAAATGAAAATATTCTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTAAAACACTTAAATATAGATCCGGTGGTATGGATGAGAAAACAATTGCTTTACTTGTTGCTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCAGGTGAATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATAT TAATATTACTTCACTACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATTGCCATATTGGTTATTCAGGTGATAGCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTGAGTATCTTAGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGCTTCCAATCAGAAGACTATACAACCTCCTAGGAAAGCTGTAGAG GAACCCCTTAATGAATAA CTGAAGTGAAGTGATGGACTCCGATTTGGAGAGTAGTAAGACGTGAAAGGAATACACTTGTGTTTAAGCACCATGGCCTTGATGATTCACTGTTGGGGAGAAGAAACAAGAAAAGTAACTGGTTGTCACCTATGAGACCCTTACGTGATTGTTAGTTAAGTTTTTATTCAAAGCAGCTGTAATTTAGTTAATAAAATAATTATGATCTATGTTGTTTGCCCAATTGAGATCCAGTTTTTTGTTGTTATTTTTAATCAATTAGGGGCAATAGTAGAATGGACAATTTCCAAGAATGATGCCTTTCAGGTCCTAGGGCCTCTGGCCTCTAGGTAACCAGTTTAAATTGGTTCAGGGTGATAACTACTTAGCACTGCCCTGGTGATTACCCAGAGATATCTATGAAAACCAGTGGCTTCCATCAAACCTTTGCCAACTCAGGTTCACAGCAGCTTTGGGCAGTTATGGCAGTATGGCATTAGCTGAGAGGTGTCTGCCACTTCTGGGTCAATGGAATAATAAATTAAGTACAGGCAGGAATTTGGTTGGGAGCATCTTGTATGATCTCCGTATGATGTGATATTGATGGAGATAGTGGTCCTCATTCTTGGGGGTTGCCATTCCCACATTCCCCCTTCAACAAACAGTGTAACAGGTCCTTCCCAGATTTAGGGTACTTTTATTGATGGATATGTTTTCCTTTTATTCACATAACCCCTTGAAACCCTGTCTTGTCCTCCTGTTACTTGCTTCTGCTGTACAAGATGTAGCACCTTTTCTCCTCTTTGAACATGGTCTAGTGACACGGTAGCACCAGTTGCAGGAAGGAGCCAGACTTGTTCTCAGAGCACTGTGTTCACACTTTTCAGCAAAAATAGCTATGGTTGTAACATATGTATTCCCTTCCTCTGATTTGAAGGCAAAAATCTACAGTGTTTCTTCACTTCTTTTCTGATCTGGGGCATGAAAAAAGCAAGATTGAAATTTGAACTATGAGTCTCCTGCATGGCAACAAAATGTGTGTCACCATCAGGCCAACAGGCCAGCCCTTGAATGGGGATTTATTACTGTTGTATCTATGTTGCATGATAAACATTCATCACCTTCCTCCTGTAGTCCTGCCTCGTACTCCCCTTCCCCTATGATTGAAAAGTAAACAAAACCCACATTTCCTATCCTGGTTAGAAGAAAATTAATGTTCTGACAGTTGTGATCGCCTGGAGTACTTTTAGACTTTTAGCATTCGTTTTTTACCTGTTTGTGGATGTGTGTTTGTATGTGCATACGTATGAGATAGGCACATGCATCTTCTGTATGGACAAAGGTGGGGTACCTACAGGAGAGCAAAGGTTAATTTTGTGCTTTTAGTAAAAACATTTAAATACAAAGTTCTTTATTGGGTGGAATTATATTTGATGCAAATATTTGATCACTTAAAACTTTTAAAACTTCTAGGTAATTTGCCACGCTTTTTGACTGCTCACCAATACCCTGTAAAAATACGTAATTCTTCCTGTTTGTGTAATAAGATATTCATATTTGTAGTTGCATTAATAATAGTTATTTCTTAGTCCATCAGATGTTCCCGTGTGCCTCTTTTATGCCAAATTGATTGTCATATTTCATGTTGGGACCAAGTAGTTTGCCCATGGCAAACCTAAATTTATGACCTGCTGAGGCCTCTCAGAAAACTGAGCATACTAGCAAGACAGCTCTTCTTGAAAAAAAAAATATGTATACACAAATATATACGTATATCTATATATACGTATGTATATACACACATGTATATTCTTCCTTGATTGTGTAGCTGTCCAAAATAATAACATATATAGAGGGAGCTGTATTCCTTTATACAAATCTGATGGCTCCTGCAGCACTTTTTCCTTCTGAAAATATTTACATTTTGCTAACCTAGTTTGTTACTTTAAAAATCAGTTTTGATGAAAGGAGGGAAAAGCAGATGGACTTGAAAAAGATCCAAGCTCCTATTAGAAAAGGTATGAAAATCTTTATAGTAAAATTTTTTATAAACTAAAGTTGTACCTTTTAATATGTAGTAAACTCTCATTTATTTGGGGTTCGCTCTTGGATCTCATCCATCCATTGTGTTCTCTTTAATGCTGCCTGCCTTTTGAGGCATTCACTGCCCTAGACAATGCCACCAGAGATAGTGGGGGAAATGCCAGATGAAACCAACTCTTGCTCTCACTAGTTGTCAGCTTCTCTGGATAAGTGACCACAGAAGCAGGAGTCCTCCTGCTTGGGCATCATTGGGCCAGTTCCTTCTCTTTAAATCAGATTTGTAATGGCTCCCAAATTCCATCACATCACATTTAAATTGCAGACAGTGTTTTGCACATCATGTATCTGTTTTGTCCCATAATATGCTTTTTACTCCCTGATCCCAGTTTCTGCTGTTGACTCTTCCATTCAGTTTTATTTATTGTGTGTTCTCACAGTGACACCATTTGTCCTTTTCTGCAACAACCTTTCCAGCTACTTTTGCCAAATTCTATTTGTCTTCTCCTTCAAAACATTCTCCTTTGCAGTTCCTCTTCATCTGTGTAGCTGCTCTTTTGTCTCTTAACTTACCATTCCTATAGTACTTTATGCATCTCTGCTTAGTTCTATTAGTTTTTTGGCCTTGCTCTTCTCCTTGATTTTAAAATTCCTTCTATAGCTAGAGCTTTTCTTTCTTTCATTCTCTCTTCCTGCAGTGTTTTGCATACATCAGAAGCTAGGTACATAAGTTAAATGATTGAGAGTTGGCTGTATTTAGATTTATCACTTTTTAATAGGGTGAGCTTGAGAGTTTTCTTTCTTTCTGTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTAATTTCACATGCTCTAAAAACCTTCAAAGGTGATTATTTTTCTCCTGGAAACTCCAGGTCCATTCTGTTTAAATCCCTAAGAATGTCAGAATTAAAATAACAGGGCTATCCCGTAATTGGAAATATTTCTTTTTTCAGGATGCTATAGTCAATTTAGTAAGTGACCACCAAATTGTTATTTGCACTAACAAAGCTCAAAACACGATAAGTTTACTCCTCCATCTCAGTAATAAAAATTAAGCTGTAATCAACCTTCTAGGTTTCTCTTGTCTTAAAATGGGTATTCAAAAATGGGGATCTGTGGTGTATGTATGGAAACACATACTCCTTAATTTACCTGTTGTTGGAAACTGGAGAAATGATTGTCGGGCAACCGTTTATTTTTTATTGTATTTTATTTGGTTGAGGGATTTTTTTATAAACAGTTTTACTTGTGTCATATTTTAAAATTACTAACTGCCATCACCTGCTGGGGTCCTTTGTTAGGTCATTTTCAGTGACTAATAGGGATAATCCAGGTAACTTTGAAGAGATGAGCAGTGAGTGACCAGGCAGTTTTTCTGCCTTTAGCTTTGACAGTTCTTAATTAAGATCATTGAAGACCAGCTTTCTCATAAATTTCTCTTTTTGAAAAAAAGAAAGCATTTGTACTAAGCTCCTCTGTAAGACAACATCTTAAATCTTAAAAGTGTTGTTATCATGACTGGTGAGAGAAGAAAACATTTTGTTTTTATTAAATGGAGCATTATTTACAAAAAGCCATTGTTGAGAATTAGATCCCACATCGTATAAATATCTATTAACCATTCTAAATAAAGAGAACTCCAGTGTTGCTATGTGCAAGATCCTCTCTTGGAGCTTTTTTGCATAGCAATTAAAGGTGTGCTATTTGTCAGTAGCCATTTTTTTGCAGTGATTTGAAGACCAAAGTTGTTTTACAGCTGTGTTACCGTTAAAGGTTTTTTTTTTTATATGTATTAAATCAATTTATCACTGTTTAAAGCTTTGAATATCTGCAATCTTTGCCAAGGTACTTTTTTATTTAAAAAAAAACATAACTTTGTAAATATTACCCTGTAATATTATATATACTTAATAAAACATTTTAAGCTATTTTGTTGGGCTATTTCTATTGCTGCTAC AGCAGACCACAAGCACATTTCTGAAAAATTTAATTTATTAATGTATTTTTAAGTTGCTTATATTCTAGGTAACAATGTAAAGAATGATTTAAAATATTAATTATGAATTTTTTGAGTATAATACCCAAATAAGCTTTTAATTAGAGCAGAGTTTTAATTAAAAGTTTTAAATCAGTCCAA In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises a deletion of the 3'-UTR. In some embodiments, the deletion comprises deletion of one or more fragments of the 3'-UTR. In some embodiments, the deletion is a complete deletion of the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises a sequence inversion in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises inversion of a fragment in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises an inversion of the entire 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises translocation of the entire 3'-UTR sequence. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises one or more nucleotide substitutions in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding CD47 comprises insertion of one or more nucleotides in the 3'-UTR.

[0085] In some embodiments, disruption in the 3'-UTR of an allele encoding CD47 results in increased expression of CD47 (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2x, at least 5x, at least 10x , at least 50-fold, at least 100-fold or more). In some embodiments, increased expression of CD47 is induced or increased by interferon gamma.

[0086] In some embodiments, the cells described herein (e.g., isolated stem cells) have a disruption in the 3'-UTR of an allele encoding histocompatibility antigen class I, G (HLA-G). Including. In some embodiments, the disruption is a homozygous modification. In some embodiments, the disruption is a heterozygous modification. "Histocompatibility antigen, class I, G (HLA-G)" belongs to HLA non-classical class I heavy chain paralogs. It is a heterodimer consisting of a heavy chain and a light chain. Heavy chains are anchored to the membrane. HLA-G is involved in immunosuppression. HLA-G is a ligand for the natural killer (NK) cell inhibitory receptor KIR2DL4. Expression of HLA-G protects expressing cells from NK cell-mediated death.

[0087] An example of the Homo sapiens HLA-G gene sequence is provided in NCBI gene ID: 3135 (SEQ ID NO: 30; corresponding to positions 29826474 to 29831130 of the Homo sapiens chromosome 6 sequence provided in NCBI accession number NC_000006.12). be done. Additionally, examples of human HLA-G transcript variants encoding different isoforms of HLA-G proteins include NCBI accession numbers NM_001363567.2 (SEQ ID NO: 7), NM_002127.6 (SEQ ID NO: 8), NM_001384280.1 (SEQ ID NO: No. 9), or NM_001384290.1 ​​(SEQ ID No. 10).

[0088] Human HLA-G gene - NCBI gene ID: 3151 (SEQ ID NO: 30); 3’-UTR underline (SEQ ID NO: 89) ATAGTAGCAGGACCACTATAGAGAGAACACTCATGTAGCAGGTCATGGAACAGTGCTAGAGCCACAGTTCAGGAGTGAGAGGGTGGTGGGGATTAAGGGGAGAAGAGGGCCTGAGGGATGAGAGGGACGGAGGGAAGGGCTGGAGGAGCAGGAGGTGAGGAAAAGGAGCAGAGGAAAGAATTCCAAAGCAGCAGAACTCTTAGGTTTAAACACATTGTTTTATAGATTTTAATACATCCATCTACAGAGCTTCGCTGGGTGTTCTTTGCAGTTGGCCTTTAATATCTTATGTGGGTCTGCCTAGAAACTAATTGTTTTTTATGTTAATCAGGTTTAAAAAATACTAAGTATTCCTAAAAAATATACACTCCACTCACATGTGGATACTTCCTAAAAACAGGCAGTGCGTGAGCACTAGTGAGGGGCATTGTGACTGCACTGAACACTTACAACTGTGAGGTGAATAAAGTTTGTGCTGGCTCCTGGTTGCAACATATAGTAACATAGTGTGGTACTTTGTCTTGAGGAGATGTCCTGGACTCACACGGAAACTTAGGGCTACGGAATGAAGGTAAATTTAAAATAAAACAAGCGGGAGTCACAGATACACTGTCTGGGAAAGTGAAACTTAAGAGCTTTGTGAGTCGTGTTGTAATGCTTTTAGATGCATTTATATACCAACAGGCCAAAGTCACATTTTTTACCGATTAGATTCCTGATCATTCAGGGGTTACCAAGGTTATGCTACCCACTATAGTTAATAAACAAAAAGCAAACTGGTCTCTATTCTATCTCATGCACTCAGGCACAACTTTTCCAGATTTAAGGGGGAAAAAAAACCCTGTCTTTACACCTACAATCCCAGGGCGAGCTCACTCTCTGGCAACAAGCTCCCTGGGGTGATTTTTCTTCTAGAAGAGTACAGGAGGACAGGCAAGGAGTGGGAGGCAGGGAGTCCAGTTCAGGGACAGGGATTCCGGGATGAAAAGTGAAGGGAGAGGGCCAGGGACCTTGCCGAGGGTTTCTCCCTGGTTTCTCAGACAGCTCCTGGGCCAAGACTCAGGGAGACACTGAGACAGAACGCTTGGCACAAGAGTAGCGGGGTCAGGGCGAAGTCCCAGGGCCTCAAGCGTGGCTCTCAGGGTCTCAGGCCCCACAGGCGGTGTATGGGTTGGGGAGGCCCCGCGTTGGGGATTCTCTCCTCCTTCTCCTAACCTGTGTCGGGTCCTTCTTCCTGGATACTCACCGGGCGGCCCCAGTTCTCACTCCCATTAGGTGACAGGTTTTTAGAGAAGCCAATCAGCGTCGCCGCGGTCCTGGTTCTAAAGTCCTCGCTCACCCACCCGGACTCATTCTCCCCAGACGCCAAGGATGGTGGTCATGGCGCCCCGAACCCTCTTCCTGCTGCTCTCGGGGGCCCTGACCCTGACCGAGACCTGGGCGGGTGAGTGCGGGGTCAGGAGGGAAACAGCCCCTGCGCGGAGGAGGGAGGGGCCGGCCCGGCGGGGGCGCAGGACTCGGCAGCCGCGCCGGGAGGAGGGTCGGGCGGGTCTCAACCCCTCCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCATGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACTCGGCGTGTCCGAGGATGGAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGGCCGGAGTATTGGGAAGAGGAGACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACCCTGCGCGGCTACTACAACCAGAGCGAGGCCAGTGAGTAACTCCGGCCCAGGGAGCAGATCACGACCCCCACCTCCATGCCCCACGGACGGCCCGGGTACTCCCGAGTCTCCGGGTCTGGGATCCACCCCGAGGCCGCGGGACCCGCCCAGACCCTCTACCTGGGAGAACCCCAAGGCGCCTTTACCAAAATCCCCGCGGGTGGGTCCGGGCGAGGGCGAGGCTCGGTGGGCGGGGCTGACCGAGGGGGTGGGGCCAGGTTCTCACACCCTCCAGTGGATGATTGGCTGCGACCTGGGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAGTATGCCTACGATGGCAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGACACTGCGGCTCAGATCTCCAAGCGCAAGTGTGAGGCGGCCAATGTGGCTGAACAAAGGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCACAGATACCTGGAGAACGGGAAGGAGATGCTGCAGCGCGCGGGTACCAGGGGCAGTGGGGCGCCTCCCTGATCTCCTGTAGACCTCTCAGCCTGGCCTAGCACAAGGAGAGGAGGAAAATGGGACCAACACTAGAATATCGCCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGATTCTGAGGGTCCGTCCTGCTCTCTGGGACAATTAAGGGATGAAGTCTCTGAGGGAGTGGAGGGGAAGACAATCCCTGGAAGACTGATCAGGGGTTCCCTTTGACCCCACAGCAGCCTTGGCACCAGGACTTTTCCCCTCAGGCCTTGTTCTCTGCCTCACACTCAATGTGTGTGGGGGTCTGACTCCAGCTCCTCTGAGTCCCTTGGCCTCCACTCAGGTCAGAACCGGAGGTCCCTGCTCCCCCGCTCAGAGACTAGAACTTTCCAAGGAATAGGAGATTATCCCAGGTGCCCGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCCCTTCCCCACCCCAGGTATCTGGTTCATTCTTAGGATGGTCACATCCAGGTGCTGCTGGAGTGTCCCATGAGAGATGCAAAGTGCTTGAATTTTCTGACTCTTCCTTTCAGACCCCCCCAAGACACACGTGACCCACCACCCTGTCTTTGACTATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGCTGAGATGGAGTAAGGAGGGAGATGGAGGCATCATGTCTGTTAGGGAAAGCAGGAGCCTCTCTGAAGACCTTTAACAGGGTCGGTGGTGAGGGCTGGGGGTCAGAGACCCTCACCTTCACCTCCTTTCCCAGAGCAGTCTTCCCTGCCCACCATCCCCATCATGGGTATCGTTGCTGGCCTGGTTGTCCTTGCAGCTGTAGTCACTGGAGCTGCGGTCGCTGCTGTGCTGTGGAGAAAGAAGAGCTCAGGTAAGGAAGGGGTGACAAGTGGGGTCTGAGTTTTCTTGTCCCACTGGGGGTTTCAAGCCCCAGGTAGAAGTGTGCCCTGCCTGGTTACTGGGAAGCACCATCCACACTCATGGGCCTACCCAGCCTGGGCCCTGTGTGCCAGCACCTTCTCTTTTGTAAAGCACCTGTGACAATGAAGGACAGATTTATTACCTTGATGATTGTAGTGATGGGGACCTGATCCCAGTAATCACAGGTCAGGAGAAGGTCCCTGGCTAAGGACAGACCTTAGGAGGGCAGTTGGTCGAGGACCCACATCTGCTTTCCTTGTTTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCGAGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTCATGGCCCTGCCACCTTTCTGGCCTCTCACAGGACATTTTCTTCCCACAGATTGA AAAGGAGGGAGCTACTCTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATCCCTGAGATCCTTGGGATCTTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTGGCCACATCTCCTGTGGTCTCTGACCAGGTGCTGTTTTTGTTCTACTCTAGGCAGTGACAGTGCCCAGGGCTCTAATGTGTCTCTCACGGCTTGTAAATGTGACACCCCGGGGGGCCTGATGTGTGGGTTGTT GAGGGGAACAGGGGACATAGCTGTGCTATGAGGTTTCTTTGACTTCAATGTATTGAGCATGTGATGGGCTGTTTAAAGTGTCACCCCTCACTGTGACTGATATGAATTTGTTCATGAATATTTTTCTG ACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTCTGTATTAAAATTAGAATCTGAGTATAAATTTACTTTTTCAAATTATTTCCAAGAGAGATTGATGGGTTAATTAAAGGAGAAGATTCCTGAAATTTGAGAGACAAAATAAATGGAAGACATGAGAACTTTCCACAGTA.

[0089] Human HLA-G transcript variant 1 - NM_001363567.2 (SEQ ID NO: 7); 3’-UTR underline (SEQ ID NO: 83) ATATAGTAACATAGTGTGGTACTTTGTCTTGAGGAGATGTCCTGGACTCACACGGAAACTTAGGGCTACGGAATGAAGACGCCAAGGATGGTGGTCATGGCGCCCCGAACCCTCTTCCTGCTGCTCTCGGGGGCCCTGACCCTGACCGAGACCTGGGCGGGCTCCCACTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCCCGGGGAGCCCGCTTCATCGCCATGGGCTACGTGGACGACACGCA GTTCGTGCGGTTCGACAGCGACTCGGCGTGTCCGAGGATGGAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGCCGGAGTATTGGGAAGAGGAGACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACCCTGCGCGGCTACTACAACCAGAGCGAGGCCAGTTCTCACACCCTCCAGTGGATGATTGGCTGCGACCTGGGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAG TATGCCTACGATGGCAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGACACTGCGGCTCAGATCTCCAAGCGCAAGTGTGAGGCGGCCAATGTGGCTGAACAAAGGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCACAGATACCTGGAGAACGGGAAGGAGATGCTGCAGCGCGCGGACCCCCCCAAGACACACGTGACCCACCACCCTGTCTTTGACTATGAGGCCACC CTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGATGGGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCCATGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGCTGAGATGGAAGCAGTCTTCCCTGCCCACCATCCCCAT CATGGGTATCGTTGCTGGCCTGGTTGTCCTTGCAGCTGTAGTCACTGGAGCTGCGGTCGCTGCTGTGCTGTGGAGAAAGAAGAGCTCAGATTGA AAAGGAGGGAGCTACTCTCAGGCTGCAATGTGAAACAGCTGCCCTGTGTGGACTGAGTGGCAAGTCCCTTTGTGACTTCAAGAACCCTGACTCCTCTTTGTGCAGAGACCAGCCCACCCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTC TGTATTAAAATTAGAATCTGAGTATAAA.

[0090] Human HLA-G transcript variant 2 - NM_002127.6 (SEQ ID NO: 8); 3’-UTR underlined (SEQ ID NO: 84) ATATAGTAACATAGTGTGGTACTTTGTCTTGAGGAGATGTCCTGGACTCACACGGAAACTTAGGGCTACGGAATGAAGTTCTCACTCCCATTAGGTGACAGGTTTTTAGAGAAGCCAATCAGCGTCGCCGCGGTCCTGGTTCTAAAGTCCTCGCTCACCCACCCGGACTCATTCTCCCAGACGCCAAGGATGGTGGTCATGGCGCCCCGAACCCTCTTCCTGCTGCTCTCGGGGGCCCTGACCCTGACCGAG ACCTGGGCGGGCTCCCACTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCATGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACTCGGCGTGTCCGAGGATGGAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGCCGGAGTATTGGGAAGAGGAGACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACC CTGCGCGGCTACTACAACCAGAGCGAGGCCAGTTCTCACACCCTCCAGTGGATGATTGGCTGCGACCTGGGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAGTATGCCTACGATGGCAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGACACTGCGGCTCAGATCTCCAAGCGCAAGTGTGAGGCGGCCAATGTGGCTGAACAAAGGAGAGCCTACCTGGAGGGCACGTGCGTGGA GTGGCTCCACAGATACCTGGAGAACGGGAAGGAGATGCTGCAGGCGCGGACCCCCCCAAGACACACGTGACCCACCACCCTGTCTTTGACTATGAGGCCACCCTGAGGTGCTGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGATGGGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTC TGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGCTGAGATGGAAGCAGTCTTCCCTGCCCACCATCCCCATCATGGGTATCGTTGCTGGCCTGGTTGTCCTTGCAGCTGTAGTCACTGGAGCTGCGGTCGCTGCTGTGCTGTGGAGAAAGAAGAGCTCAGATTGA AAAGGAGGGAGCTACTCTCAGGCTGCAATGTGAAACAGCTGCCCTGTGTGGACTGAGTGGCAAGTCCCTTTGTGACTTCAAGAACCCTGACTCCTCTTTGTGCAGAGACCAGCCCACCCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTC TGTATTAAAATTAGAATCTGAGTATAAA.

[0091] Human HLA-G transcript variant 3 - NM_001384280.1 (SEQ ID NO: 9); 3’-UTR underline (SEQ ID NO: 85) ATAGTAGCAGGACCACTATAGAGAGAACACTCATGTAGCAGGTCATGGAACAGTGCTAGAGCCACAGTTCAGGAATGTCCTGGACTCACACGGAAACTTAGGGCTACGGAATGAAGACGCCAAGGATGGTGGTCATGGCGCCCCGAACCCTCTTCCTGCTGCTCTCGGGGGCCCTGACCCTGACCGAGACCTGGGCGGGCTCCCACTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCGGGGA GCCCCGCTTCATCGCCATGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACTCGGCGTGTCCGAGGATGGAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGGCCGGAGTATTGGGAAGAGGAGACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACCCTGCGCGGCTACTACAACCAGAGCGAGGCCAGTTCTCACACCCTCCAGTGGATGATTGGCTGCGACCTG GGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAGTATGCCTACGATGGCAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGACACTGCGGCTCAGATCTCCAAGCGCA CGTGACCCACCACCCTGTCTTTGACTATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGATGGGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGC TGAGATGGAAGCAGTCTTCCCTGCCCACCATCCCCATCATGGGTATCGTTGCTGGCCTGGTTGTCCTTGCAGCTGTAGTCACTGGAGCTGCGGTCGCTGCTGTGCTGTGAGAAAGAAGAGCTCAGATTGA AAAGGAGGGAGCTACTCTCAGGCTGCAATGTGAAACAGCTGCCCTGTGTGGACTGAGTGGCAAGTCCCTTTGTGACTTCAAGAACCCTGACTCCTCTTTGTGCAGAGACCAGCCCACCCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTC TGTATTAAAATTAGAATCTGAGTATAAA.

[0092] Human HLA-G transcript variant 4-NM_001384290.1 ​​(SEQ ID NO: 10); 3’-UTR underline (SEQ ID NO: 86) ATTCTCCCCAGACGCCAAGGATGGTGGTCATGGCGCCCCGAACCCTCTTCCTGCTGCTCTCGGGGGCCCTGACCCTGACCGAGACCTGGGCGGGCTCCCACTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCATGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACTCGGCGTGTCCGAGGATGGAGCCGCGGGCGCCGTGGGTGGA GCAGGAGGGGCCGGAGTATTGGGAAGAGGAGACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACCCTGCGCGGCTACTACAACCAGAGCGAGGCCAGTTCTCACACCCTCCAGTGGATGATTGGCTGCGACCTGGGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAGTATGCCTACGATGGCAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGA CACTGCGGCTCAGATCTCCAAGCGCAAGTGTGAGGCGGCCAATGTGGCTGAACAAAGGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCACAGATACCTGGAGAACGGGAAGGAGATGCTGCAGCGGCGCGGACCCCCCCAAGACACACGTGACCCACCACCCTGTCTTTGACTATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGGATG GGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGCTGAGATGGAAGCAGTCTTCCCTGCCCACCATCCCCATCATGGGTATCGTTGCTGGCCTGGTTGTCCTTGCAGCTGTAGTCACTGGAGCTGCGGTCGCTG CTGTGCTGTGGAGAAAGAAGAGCTCAGATTGA AAAGGAGGGAGCTACTCTCAGGCTGCAATGTGAAACAGCTGCCCTGTGTGGACTGAGTGGCAAGTCCCTTTGTGACTTCAAGAACCCTGACTCCTCTTTGTGCAGAGACCAGCCCACCCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTC TGTATTAAAATTAGAATCTGAGTATAAA.

[0093] In some embodiments, the disruption in the 3'-UTR of the allele encoding HLA-G comprises a deletion of the 3'-UTR. In some embodiments, the deletion comprises deletion of one or more fragments of the 3'-UTR. In some embodiments, the deletion is a complete deletion of the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding HLA-G comprises a sequence inversion in the 3'-UTR. In some embodiments, disruption in the 3'-UTR of an allele encoding HLA-G comprises inversion of a fragment in the 3'-UTR. In some embodiments, disruption in the 3'-UTR of an allele encoding HLA-G comprises an inversion of the entire 3'-UTR. In some embodiments, disruption in the 3'-UTR of an allele encoding HLA-G comprises translocation of the entire 3'-UTR sequence. In some embodiments, the disruption in the 3'-UTR of the allele encoding HLA-G comprises substitution of one or more nucleotides in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding HLA-G comprises insertion of one or more nucleotides in the 3'-UTR.

[0094] In some embodiments, the present disclosure describes the HLA-G 3'-UTR at nucleotide positions +3001, +3003, +3010, +3027, +3032, +3035, +3052, +3092, +3111, +3121 , +3142, +3177, +3183, +3187, +3196, and +3227 are deleted or substituted with alternative nucleotides. In some embodiments, the present disclosure provides that one or both copies of the HLA-G 3'-UTR of the cell is +3003T, +3010G, +3010C, +3035C, +3142C, +3142G, +3187G, Cells containing any one or a combination of +3187A, +3196C, +3196G, +3227G, +3227A are contemplated. In some embodiments, the present disclosure describes the HLA-G 3'-UTR nucleotide positions +3001, +3003, +3010, +3027, +3032, +3035, +3052, +3092, +3111, +3121 , +3142, +3177, +3183, +3187, +3196, and +3227 are deleted or replaced with alternative nucleotides, and microRNAs (e.g., miR-133A, miR-148A, miR-148B, miR -152, miR-548q and / or miR-628-5p) are unable to bind or have significantly reduced binding to the 3'-UTR of HLA-G RNA transcripts. The cells that are present are planned. In some embodiments, the present disclosure provides that at least 5, 8, 10, 12, 14, 20 contiguous nucleotides starting from and including position +2961 of the HLA-G 3'-UTR are missing. Cells that have lost and / or have inserted at least 5, 8, 10, 12, 14, 20 nucleotides at position +2961 are contemplated. In some embodiments, the present disclosure provides at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Contemplated are cells in which at least one insertion, deletion, substitution, translocation has been introduced into a nucleic acid sequence that corresponds to a sequence that is %, 99% or 100% identical. In some embodiments, the present disclosure provides that at least 1, 3, 5, 8, 10, 12 or 14 nucleotides of SEQ ID NO: 75 (ATTTGTTCATGCCT) are not present in the nucleic acid (e.g., have been deleted from the nucleic acid). ), at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74 Contains a nucleic acid comprising a sequence (e.g., SEQ ID NO: 74 and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical). In some embodiments, the cell has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 74. , 99% or 100% identical, including nucleic acids in which a G is present at the position corresponding to position 120 of the sequence. In some embodiments, the cell has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 74. , 99% or 100% identical, including nucleic acids in which a C is present at the position corresponding to position 252 of the sequence. In some embodiments, the cell has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 74. , 99% or 100% identical, including nucleic acids in which a G is present at the position corresponding to position 297 of the sequence. In some embodiments, the cell has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 74. , 99% or 100% identical, including nucleic acids in which a G is present at the position corresponding to position 306 of the sequence. See, for example, Poras et al., 2017, PLOS One, DOI:10.1371 / journal.pone.0169032; Schwich et al., 2019, Scientific Reports, 9:5407. In some embodiments, the cell is heterozygous for any one or combination of the above genetic elements listed in this paragraph. In some embodiments, the cell is homozygous for any one or combination of the above genetic elements listed in this paragraph.

[0095] Sequence number 74 ATTGAAAAGGAGGGAGCTACTCTCAGGCTGCAATGTGAAACAGCTGCCCTGTGTGGACTGAGTGGCAAGATTTGTTCATGCCTTCCCTTTGTGACTTCAAGAACCCTGACTTCTCTTTCTGCAGAGACCAGCCCACCCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCCTCAAATTTGTGGTGCACT GAGCTATAACTTACTTCTGTATTAAAATTAGAATCTGAGTATAAATTTACTTTTTCAAATTATTTCCAAGAGAGATTGATGGGTTAATTAAAGGAGAAGATTCCTGAAATTTGAGAGACAAAATAAATGGAAGAC.

[0096] In some embodiments, disruption in the 3'-UTR of allelic HLA-G results in increased expression of HLA-G (e.g., in cells, isolated stem cells, and cells differentiated from isolated stem cells). , at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 5 times, at least 10x, at least 50x, at least 100x or more). In some embodiments, increased expression of HLA-G is induced or increased by interferon gamma.

[0097] In some embodiments, the cells described herein (eg, isolated stem cells) do not contain additional exogenous expression of any factors (eg, proteins or RNA). In some embodiments, an isolated cell described herein (eg, a stem cell) does not contain an insertion of an exogenous nucleotide sequence anywhere in its genome. In some embodiments, the present disclosure describes the 3'-UTR of two or more alleles in the genome of a cell, e.g., two or more of an allele encoding any of PD-L1, CD47, or HLA-G. An isolated cell (eg, a stem cell) is provided that includes a disruption in one or more 3'-UTRs.

[0098] In some embodiments, a cell described herein (eg, an isolated stem cell) comprises a disruption in the 3'-UTR of an allele encoding PDL2. In some embodiments, the disruption is a homozygous modification. In some embodiments, the disruption is a heterozygous modification. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises a deletion of the 3'-UTR. In some embodiments, the deletion comprises deletion of one or more fragments of the 3'-UTR. In some embodiments, the deletion is a complete deletion of the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises a sequence inversion in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises inversion of a fragment in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises an inversion of the entire 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises translocation of the entire 3'-UTR sequence. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises one or more nucleotide substitutions in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding PDL2 comprises insertion of one or more nucleotides in the 3'-UTR. In some embodiments, the 3'-UTR of PDL2 referred to herein comprises at least 75%, 80%, 85%, 90% of the nucleotide sequence of SEQ ID NO: 76, and 90-96, or a portion thereof. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0099] Sequence number 76 (accession number NM_025239) GGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAACAATTGGCAGAGCTCAGAATTCAAGCGATCGCCACAAAGAGGGAAGTGAACAGTGCTATCTGAACCTGTGGTCTTGGGAGCCAGGGTGACCTGATATGACATCTAAAGAAGCTTCTGGACTCTGAACAAGAATTCGGTGGCCTGCAGAGCTTGCCATTTGCACTTTTCAAATGCCTTTGGATGACCCAGC ACTTTAATCTGAAACCTGCAACAAGACTAGCCAACACCTGGCCATGAAACTTGCCCCTTCACTGATCTGGACTCACCTCTGGAGCCTATGGCTTTAAGCAAGCACTACTTTACAGAATTACCCCACTGG TGGGTTGCCAATAGAGTTATTTTTTATCTATAGCTTCCTCTGGGTACTAGAAGAGGCTATTGAGACTATGAGCTCACAGACAGGGCTTCGCACAAACTCAAATCATAATTGACATGTTTTATGGATTACTGGAATCTTGATAGCATAATGAAGTTGTTCTAATTAACAGAGAGCATTTAAATATACACTAAGTGCACAAATTGTGGAGTAAAGTCATCAAGCTCTGTTTTTGAGGTCTAAGTCACAAAGCATTTGTTTTAACC TGTAATGGCACCATGTTTAATGGTGGTTTTTTTTGAACTACATCTTTCCTTTAAAAATTATTGGTTTCTTTTTATTTGTTTTTACCTTAGAAATCAATTATATACAGTCAAAAATATTTGATATGCTCATACGTTGTATCTGCAGCAATTTCAGATAAGTAGCTAAAATGGCCAAAGCCCCAAACTAAGCCTCCTTTTCTGGCCCTCAATATGACTTTAAATTTGACTTTTCAGTGCCTCAGTTTGCACATCTGTAATACAG CAATGCTAAGTAGTCAAGGCCTTTGATAATTGGCACTATGGAAATCCTGCAAGATCCCACTACATATGTGTGGAGCAGAAGGGTAACTCGGCTACAGTAACAGCTTAATTTTGTTAAATTTGTTCTTTATAC TGGAGCCATGAAGCTCAGAGCATTAGCTGACCCTTGAACTATTCAAATGGGCACATTAGCTAGTATAACAGACTTACATAGGTGGGCCTAAAGCAAGCTCCTTAACTGAGCAAAATTTGGGGCTTATGAGAAT GAAAGGGTGTGAAATTGACTAACAGACAAATCATACATCTCAGTTTCTCAATTCTCATGTAAATCAGAGAATGCCTTTAAAGAATAAAACTCAATTGTTATTCTTCAACGTTCTTTATATATTCTACTTTTGGGTAACGCGTAAGCGGCCGCGGCATCTAGATTCGAAGAAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGG.

[0100] In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 581-603 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 362-387 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 394-416 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 699-723 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 1333-1353 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 686-709 of SEQ ID NO:76. In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 764-785 of SEQ ID NO:76. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 90 (AAGAGGCTATTGAGACTATGAGC) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 91 (AAGCACTACTGCACTTTACAGAATTA) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 92 (TGGATCCTGGACCCACAGAATTC) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, this disclosure contemplates cells in which SEQ ID NO: 93 (GAGAGCATTTAAATATACACTAAGT) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 94 (GAAATTGACTAACAGACAAAT) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 95 (GTTCTAATTAACAGAGCATTTA) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 96 (GGTCTAAGTCACAAAGCATTTG) of the PDL2 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the cell has one or more nucleotide deletions, insertions, and / or insertions in any one of 76 or 90-96 such that the endogenous microRNA has reduced or abolished binding in the cell. or include substitutions. In some embodiments, the endogenous microRNA is any one or more of miR-BHRF1-2-5p, miR-BART1-5p, miR-BART7-3p, and / or miR-BART14-3p. .

[0101] In some embodiments, the present disclosure provides a , 21, 22, 23 or 24, or all nucleotides are deleted from any one of 76 and 90-96 of SEQ ID NO: PDL2 3'-UTR. In some embodiments, the present disclosure provides a , 21, 22, 23 or 24 or all nucleotides are substituted in any one of SEQ ID NOs: 76 and 90-96 of the PDL2 3'-UTR. In some embodiments, the present disclosure provides a , 21, 22, 23 or 24 or all nucleotides inserted into SEQ ID NOs: 76 and 90-96 of the PDL2 3'-UTR. See, eg, Cristino, 2019, Blood, 134(25):2261-2270, which is incorporated herein by reference in its entirety. Because the sequences SEQ ID NOs: 76 and 90-96 of the PDL2 3'-UTR are derived from naturally occurring nucleotide sequences in cells, the nucleic acids in cells may have some differences compared to these reference sequences, e.g. Please note that there is a possibility of having polymorphisms). Accordingly, the present disclosure provides that cells have one, two, three, four, five, , or nucleotide sequences having six or fewer nucleotide differences. In some embodiments, the cell is heterozygous for any one or combination of the above genetic elements listed in this paragraph. In some embodiments, the cell is homozygous for any one or combination of the above genetic elements listed in this paragraph.

[0102] In some embodiments, disruption in the 3'-UTR of an allele encoding PDL2 results in increased expression of PDL2 (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2x, at least 5x, at least 10x , at least 50-fold, at least 100-fold or more). In some embodiments, increased expression of PDL2 is induced or increased by interferon gamma.

[0103] In some embodiments, a cell described herein (eg, an isolated stem cell) comprises a disruption in the 3'-UTR of an allele encoding IL-10. In some embodiments, the disruption is a homozygous modification. In some embodiments, the disruption is a heterozygous modification. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises a deletion of the 3'-UTR. In some embodiments, the deletion comprises deletion of one or more fragments of the 3'-UTR. In some embodiments, the deletion is a complete deletion of the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises a sequence inversion in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises inversion of a fragment in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises an inversion of the entire 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises translocation of the entire 3'-UTR sequence. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises one or more nucleotide substitutions in the 3'-UTR. In some embodiments, the disruption in the 3'-UTR of the allele encoding IL-10 comprises insertion of one or more nucleotides in the 3'-UTR. In some embodiments, the 3'-UTR of IL-10 referred to herein is at least 75%, 80%, 85%, Includes sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0104] Sequence number 77 GACATCAGGGTGGCGACTCTATAGACTCTAGGACATAAATTAGAGGTCTCCAAAATCGGATCTGGGGCTCTGGGATAGCTGACCCAGCCCCTTGAGAAACCTTATTGTACCTCTCTTATAGAATATTTATTACCTCTGATACCTCAACCCCCATTTCTATTTATTTACTGAGCTTCTCTGTGAACGATTTAGAAAGAAGCCCAATATTATAATTTTTTTCAATATTTATTATTTTCACCTGTTTTTAAGCTGTTTCCATAGGG TGACACACTATGGTATTTGAGTTTTAAGATAAATTATAAGTTACATAAGGGAGGAAAAAAAATGTTCTTTGGGGAGCCAACAGAAGCTTCCATTCCAAGCCTGACCACGCTTTCTAGCTGTTGAGCTGTTTTCCCTGACCTCCCTCTAATTTATCTTGTCTCTGGGCTTGGGGCTTCCTAACTGCTACAAATACTCTTAGGAAGAGAAACCAGGGAGCCCCTTTGATGATTAATTCACCTTCCAGTGTCTCGG AGGGATTCCCCTAACCTCATTCCCCAACCACTTCATTCTTGAAAGCTGTGGCCAGCTTGTTATTTATAACAACCTAAATTTGGTTCTAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGA GAGGCTGAGGCAAGAGAATTGCTTGAACCCAGGAGATGGAAGTTGCAGTGAGCTGATATCATGCCCCTGTACTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTCAAAAAATAAAAATAAAAATAAATTTGGTTCTAATAGAACTCAGTTTTAACTAGAATTTATTCAATTCCTCTGGGAATGTTACATTGTTTGTCTGTCTTCATAGCAGATTTTAATTTTGAATAAATAAATGTATCTTATTCACATC.

[0105] In some embodiments, the cell comprises a nucleotide disruption corresponding to nucleotides 125-147 of SEQ ID NO:77. In some embodiments, the present disclosure contemplates cells in which SEQ ID NO: 97 (ATTTATTACCTCTGATACCTCAA) of the IL-10 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the present disclosure contemplates cells in which the TACCTCA of the IL-10 3'-UTR comprises one or more nucleotide deletions, insertions, and / or substitutions. In some embodiments, the cell has one or more nucleotide deletions, insertions, and insertions in any one of 77, 97, or TACCTCA such that the endogenous microRNA has reduced or abolished binding in the cell. / or include substitutions. In some embodiments, the endogenous microRNA is any one or more of let-7b, let-7c, or let-7f.

[0106] In some embodiments, the present disclosure provides a , 21, 22, 23 or 24, or all nucleotides are deleted from any one of SEQ ID NO: 77, 97, or TACCTCA of the IL-10 3'-UTR. In some embodiments, the present disclosure provides a , 21, 22, 23 or 24 or all nucleotides are substituted in any one of SEQ ID NO: 77, 97, or TACCTCA of the IL-10 3'-UTR. In some embodiments, the present disclosure provides a , 21, 22, 23 or 24 or all nucleotides are inserted into any one of SEQ ID NO: 77, 97, or TACCTCA of the IL-10 3'-UTR. See, eg, Swaminathan et al., 2012, J. Immunol., 188(12):6238-6246, herein incorporated by reference in its entirety. Please note that the sequences of IL-10 3'-UTR SEQ ID NO: 77, 97, or TACCTCA are derived from naturally occurring nucleotide sequences in cells, so the nucleic acids in cells are , may have some differences (eg, polymorphisms). Accordingly, the present disclosure provides that the present disclosure provides that the cell has 1, 2, 3, 4, 5, or 1, 2, 3, 4, 5, or It is contemplated that nucleotide sequences with no more than six nucleotide differences may be included. In some embodiments, the cell is heterozygous for any one or combination of the above genetic elements listed in this paragraph. In some embodiments, the cell is homozygous for any one or combination of the above genetic elements listed in this paragraph.

[0107] In some embodiments, disruption in the 3'-UTR of an allele encoding IL-10 increases the expression of IL-10 in a cell, an isolated stem cell, or a cell differentiated from an isolated stem cell. (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 5 times , at least 10-fold, at least 50-fold, at least 100-fold or more). In some embodiments, increased expression of IL-10 is induced or increased by interferon gamma.

[0108] In some embodiments, the cells described herein (eg, isolated stem cells) further comprise exogenous expression of one or more immunosuppressive factors. In some embodiments, a cell described herein (e.g., an isolated stem cell) has inserted into its genome a polynucleotide that includes a nucleotide sequence encoding one or more immunosuppressive factors. Including further. Non-limiting examples of one or more immunosuppressive factors for exogenous expression include CD47, PDL1, PDL2, CTLA-4, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL -35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and SERPINB9. Nucleotide sequences encoding immunosuppressive factors (eg, CD47, PDL1, CTLA-4, HLA-C, HLA-E, HLA-G, C1 inhibitor, or IL-35) are known in the art. In some embodiments, the cells contain a polynucleotide sequence encoding one or more of TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, and / or IFNγR1 d39. Contains nucleotide insertions. In some embodiments, the nucleotide sequence encoding one or more immunosuppressive factors that is inserted into the genome of a cell described herein (e.g., an isolated stem cell) is modified (e.g., , codon-optimized).

[0109] In some embodiments, the one or more immunosuppressive factors for exogenous expression are CD47, PDL1, and / or CTLA-4. Non-limiting examples of nucleotide sequences encoding different isoforms of CD47 protein include NCBI accession numbers NM_001777.4 (SEQ ID NO: 4), NM_198793.3 (SEQ ID NO: 5), or NM_001382306.1 (SEQ ID NO: 6). shown. Non-limiting examples of nucleotide sequences encoding different isoforms of PDL1 protein include NCBI accession numbers NM_014143.4 (SEQ ID NO: 1), NM_001267706.2 (SEQ ID NO: 2), or NM_001314029.2 (SEQ ID NO: 3). listed. Non-limiting examples of human nucleotide sequences encoding different isoforms of HLA-G proteins include NCBI accession numbers NM_001363567.2 (SEQ ID NO: 7), NM_002127.6 (SEQ ID NO: 8), NM_001384280.1 (SEQ ID NO: 9). ), or NM_001384290.1 ​​(SEQ ID NO: 10). Non-limiting examples of human nucleotide sequences encoding different isoforms of CTLA-4 protein are shown in NCBI accession numbers NM_001037631.3 (SEQ ID NO: 11) or NM_005214.5 (SEQ ID NO: 12).

[0110] Non-limiting examples of amino acid sequences of different isoforms of the CD47 protein are shown in NCBI accession numbers NP_001369235.1 (SEQ ID NO: 49), NP_001768.1 (SEQ ID NO: 50), or NP_942088.1 (SEQ ID NO: 51). are. Non-limiting examples of amino acid sequences of different isoforms of PDL1 protein are shown in NCBI accession numbers NP_001254635.1 (SEQ ID NO: 52), NP_001300958.1 (SEQ ID NO: 53), or NP_054862.1 (SEQ ID NO: 54) . Non-limiting examples of amino acid sequences of different isoforms of CTLA-4 protein are shown in NCBI accession number NP_001032720.1 (SEQ ID NO: 55) or NP_005205.2 (SEQ ID NO: 56).

[0111] In some embodiments, an isolated cell described herein (e.g., an isolated stem cell) has at least 75% of the amino acid sequence of any one of SEQ ID NOs: 49-56, or a fragment thereof. Insertion of a foreign polynucleotide comprising a nucleotide sequence encoding a polypeptide that is (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical include. In some embodiments, an isolated cell described herein (e.g., an isolated stem cell) comprises an amino acid sequence of any one of SEQ ID NOs: 49-56, or a fragment or variant thereof. Involves the insertion of an exogenous polynucleotide comprising a nucleotide sequence encoding a polypeptide.

[0112] In some embodiments, the isolated cells described herein (e.g., isolated stem cells) have one or more immunosuppressive factors (e.g., CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, C1 inhibitors, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9). Insertion of an exogenous polynucleotide comprising an encoding nucleotide sequence into the disrupted 3'-UTR locus of an endogenous immunosuppressive factor gene (e.g., PDL1, CD47 or HLA-G) within a cell (e.g., a stem cell) further including. In some embodiments, one or more immunosuppressive factors (e.g., CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL-35, DUX4, An exogenous polynucleotide sequence encoding an endogenous immunosuppressive factor gene (e.g., PDL1, CD47) in a cell encodes an exogenous polynucleotide sequence encoding an endogenous immunosuppressive factor gene (e.g., PDL1, CD47, or HLA-G) into the disrupted 3'-UTR locus, RNA (eg, mRNA) containing the coding sequences for both immunosuppressive factors is obtained. In some embodiments, one or more immunosuppressive factors (e.g., CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL-35, DUX4, Exogenous polynucleotides encoding endogenous immunosuppressive factor genes (e.g., PDL1, CD47 or HLA-G) into the disrupted 3'-UTR locus compared to cells of the same cell type lacking the 3'-UTR locus of the exogenous polynucleotide and disrupted endogenous immunosuppressive factor genes. increased levels of cellular expression of exogenous polynucleotides and endogenous immunosuppressive factor genes (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, an increase of at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold or more).

[0113] In some embodiments, the isolated cells described herein (e.g., stem cells) have one or more immunosuppressive factors (e.g., CD47, CTLA-4, PDL1, PDL2, HLA-C , HLA-E, HLA-G, C1 inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9). into a safe harbor locus (eg, the AAVS1 locus). As used herein, a "safe harbor locus" refers to a genomic locus where a gene or other genetic element can be safely inserted and expressed. Genes or genetic elements randomly inserted into the host genome may interact with host genes or host genetic elements in unpredictable ways. A safe harbor locus is a known site for safely inserting a foreign gene or genetic element that ensures proper expression and function of the foreign gene or genetic element without significantly compromising the health of the cell.

[0114] In some embodiments, any of the isolated cells disclosed herein (eg, any of the stem cells disclosed herein) comprises a "safety switch." In some embodiments, the safety switch is a nucleic acid construct that encodes a switch protein that inducibly causes cell death or stops cell proliferation. In some embodiments, the safety switch is inserted into the genome of the engineered cell at a defined specific target locus (e.g., a safe harbor locus), typically on both alleles of the target locus. be done. In some embodiments, the target locus is a safe harbor locus, such as ActB or CLYBL. In some embodiments, the switch protein is activated by contacting it with an effective amount of a clinically acceptable orthologous small molecule. In some embodiments, when activated, the safety switch halts cell proliferation, in some embodiments by activating apoptosis of the cell. In some embodiments, the switch protein comprises herpes-simplex-thymidine kinase. In some embodiments, the switch protein is a human caspase protein, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, etc. Including. In certain embodiments, the protein is human caspase-9. In some embodiments, the caspase protein is fused to a sequence that provides chemically induced dimerization (CID), with dimerization occurring only in the presence of an ortholog activating agent. One or more CID domains can be fused to a caspase protein, eg, two different CID domains can be fused to a caspase protein. In some embodiments, the CID domain is the dimerization domain of the FKBP or FRB (FKBP-rapamycin binding) domain of mTOR, which is activated using a rapamycin analog. In some embodiments, the safety switch is any of the safety switches described in International Application Publication No. WO2021 / 173449 and Jones et al., 2014, Frontiers in Pharmacology, 5(254): pages 1-8. , each of which is incorporated herein in its entirety.

[0115] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocation, inversion, or substitution) and further comprises insertion of a polynucleotide encoding CD47 in the disrupted 3'-UTR locus of PDL1. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CD47 at safe harbor loci.

[0116] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocation, inversion, or substitution), and further comprises insertion of a polynucleotide encoding CTLA-4 in the disrupted 3'-UTR locus of PDL1. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CTLA-4 at safe harbor loci.

[0117] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocation, inversion, or substitution), and further comprises insertion of a polynucleotide encoding PDL1 at the disrupted 3'-UTR locus of PDL1. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding PDL1. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding PDL1 at safe harbor loci.

[0118] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. (translocation, inversion, or substitution), and further comprises insertion of a polynucleotide encoding CD47 at the disrupted 3'-UTR locus of CD47. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CD47 at safe harbor loci.

[0119] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. translocation, inversion, or substitution), and further comprises insertion of a polynucleotide encoding CTLA-4 at the disrupted 3'-UTR locus of CD47. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CTLA-4 at safe harbor loci.

[0120] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. translocation, inversion, or substitution) and further comprises insertion of a polynucleotide encoding PDL1 in the disrupted 3'-UTR locus of CD47. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, insertion, etc.) in the 3'-UTR of an allele encoding CD47. translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding PDL1 at safe harbor loci.

[0121] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, insertions, translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CD47 in the disrupted 3'-UTR locus of HLA-G. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, (insertions, translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CD47 at safe harbor loci.

[0122] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, insertions, translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CTLA-4 in the disrupted 3'-UTR locus of HLA-G. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, (insertions, translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding CTLA-4 at safe harbor loci.

[0123] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, insertions, translocations, inversions, or substitutions), and further comprises insertions of polynucleotides encoding PDL1 in the disrupted 3'-UTR locus of HLA-G. In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., a deletion, (insertions, translocations, inversions, or substitutions), and further includes insertions of polynucleotides encoding PDL1 at safe harbor loci.

[0124] In some embodiments, any of the isolated cells described herein (e.g., any of the stem cells) have MHC-I and MHC-II human leukocyte antigens against wild-type cells of the same cell type. (HLA) expression (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% decreased) further including. The major histocompatibility complex (MHC) is a locus on human Chr.6p21 that encodes a highly polymorphic gene family of surface molecules that determine donor compatibility during organ transplantation. MHC class I (MHC-I) and MHC class II (MHCII) play an important role in activating adaptive immune responses by presenting antigens to T lymphocytes. Humans have three classical MHC-Ia molecules (HLA-A, HLA-B, and HLA-C) that are essential for the detection and elimination of viruses, cancer cells, and transplanted cells. Additionally, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F, HLA-G) that have immunomodulatory functions. While MHC serves important functions, it can also contribute to immune rejection in certain situations, such as in cell-based transplantation therapies.

[0125] The MHC-I molecule is composed of an MHC-encoded heavy chain and an invariant subunit β2-microglobulin (B2M). Antigen-derived peptides are presented to CD8 T cells, which have antigen-specific T cell receptors, by MHC-I-B2M complexes on the cell surface. Most of the peptides are produced from the degradation of cytoplasmic proteins by specialized proteasomes or immunoproteasomes, which are optimized to generate MHC class I peptides and include some IFN-γ inducible subunits. Unlike MHC-II, which is primarily present on antigen-presenting cells, MHC-Ia is ubiquitously expressed on almost all nucleated cells (e.g., Pamer et al., Annu Rev Immunol, incorporated herein by reference). (1998) 16:323-358.) Both MHC-I and MHC-II genes are highly inducible by IFN-γ stimulation.

[0126] In certain embodiments, reducing the expression of MHC-I and MHC-II HLAs is accomplished by targeting individual HLAs (e.g., disrupting genes encoding HLA-A, HLA-B, and / or HLA-C). targeting transcriptional regulators of HLA expression (e.g., disrupted genes encoding NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C and / or IRF-1) ), or by blocking surface transport of MHC class I molecules (eg, disrupted genes encoding B2M and / or TAP1), and / or by targeting HLA-Razor. In certain embodiments, the genes encoding HLA-A and HLA-B are individually disrupted and the gene encoding HLA-C is not disrupted. In certain embodiments, the genes encoding HLA-A and HLA-C are individually disrupted, and the gene encoding HLA-B is not disrupted. In certain embodiments, the genes encoding HLA-B and HLA-C are individually disrupted, and the gene encoding HLA-A is not disrupted.

[0127] In some embodiments, the reduced expression of MHC-I human leukocyte antigen is due to a disruption in the allele encoding β-2 microglobulin (B2M). Thus, in some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption (e.g., deletion, translocation, inversion, etc.) in the allele encoding B2M. , or substitution). In some embodiments, the disruption (e.g., deletion, insertion, translocation, inversion, or substitution) in the allele encoding B2M is at least 10%, at least 20%, at least 30%, at least 40%, results in a reduction in B2M expression of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, none of the cells disclosed herein contain a disruption of the allele encoding B2M.

[0128] In some embodiments, the reduced expression of MHC-II human leukocyte antigen is due to a disruption in an allele encoding class II major histocompatibility complex transactivator (CIITA). Thus, in some embodiments, any of the isolated cells described herein (e.g., stem cells) contain a disruption (e.g., deletion, insertion, translocation, etc.) in an allele encoding CIITA. (inversion, or substitution). In some embodiments, the disruption (e.g., deletion, insertion, translocation, inversion, or substitution) in the allele encoding CIITA is at least 10%, at least 20%, at least 30%, at least 40%, resulting in a reduction in CIITA expression of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, any of the cells disclosed herein does not contain a disruption of an allele encoding CIITA.

[0129] In some embodiments, any of the isolated cells described herein (e.g., stem cells) have a disruption in the allele encoding B2M (e.g., deletion, insertion, translocation, inversion). , or substitutions), and disruptions (eg, deletions, insertions, translocations, inversions, or substitutions) in alleles encoding CIITA.

[0130] In some embodiments, any of the isolated cells described herein (e.g., stem cells) are B2M, CIITA, HLA-A, HLA-B, HLA-C, RFX-ANK, NFY -A, NLRC5, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAPI, GITR, 4-1BB, CD28, B7-1, CD47, B7 -2, 0X40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2 , CD58, CD2, HELIOS, IDO1, TRAC, TRB, NFY-A, CCR5, F3, CD142, MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D, PDGFRa, OLIG2, and / or GFAP Including disruptions (eg, deletions, insertions, translocations, inversions, or substitutions) in any one or more of the genes.

[0131] In some embodiments, any of the isolated cells described herein (e.g., stem cells) contain MHC-I and MHC-II human leukocytes compared to wild-type stem cells of the same cell type. Does not include decreased expression of antigen (HLA). In some embodiments, any of the cells described herein (e.g., isolated stem cells) have a disruption (e.g., deletion, translocation) in an allele encoding B2M or an allele encoding CIITA. loci, inversions, or substitutions).

[0132] In some embodiments, the cells described herein (eg, isolated stem cells) are negative for A antigen and negative for B antigen. In some embodiments, the cells described herein are negative for A antigen. In some embodiments, the cells described herein are negative for B antigen. In some embodiments, the cells described herein (eg, isolated stem cells) are negative for Rh antigen. In some embodiments, the cells described herein (e.g., isolated stem cells) are negative for A antigen, negative for B antigen, and negative for Rh antigen. It is. As used herein, "A antigen" refers to a histoblood group antigen produced by 3α-N-acetylgalactosaminyltransferase and expressed as a cell surface antigen. As used herein, "B antigen" refers to a histoblood group antigen produced by 3α-galactosaminyltransferase and expressed as a cell surface antigen. In some embodiments, the cell comprises a disruption of the ABO gene. In some embodiments, the cell comprises a disruption of the ABO gene such that the cell has reduced or eliminated levels of A and B antigens. In some embodiments, the cell comprises a disruption of the FUT1 gene. In some embodiments, the cell comprises a disruption of the FUT1 gene, resulting in reduced or eliminated expression of galactoside 2-alpha-L-fucosyltransferase 1. As used herein, "Rh antigen" refers to a highly immunogenic antigen encoded by two highly polymorphic genes, RHD and RHCE. Rh antigen protein is a transmembrane protein. In some embodiments, the cell comprises a disruption of the RHAG gene. In some embodiments, the cell comprises a disruption of the RHAG gene such that the cell has reduced or absent levels of Rh-related glycoproteins. In some embodiments, the cells contain Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNS antigen U, and MNS antigen Rh protein antigen expression is reduced or eliminated selected from the group consisting of S.

[0133] In some embodiments, the cells described herein (eg, isolated stem cells) are embryonic stem cells. In some embodiments, the cells described herein (eg, isolated stem cells) are embryonic germ stem cells (EGSCs). In some embodiments, the cells described herein (eg, isolated stem cells) are pluripotent stem cells. In some embodiments, the cells described herein (eg, isolated stem cells) are induced pluripotent stem cells. In some embodiments, the cells described herein (eg, isolated stem cells) are reprogrammed stem cells derived from somatic cells. In some embodiments, the cells described herein (e.g., isolated stem cells) are human stem cells (e.g., human embryonic stem cells, or human pluripotent stem cells, such as human induced pluripotent stem cells). It is.

[0134] As used herein, the term "stem cell" can refer to cells (e.g., vertebrate stem cells, mammalian stem cells) that have both the capacity for self-renewal and the capacity to generate differentiated cell types (Morrison et al. , (1997) Cell 88:287-298). In the context of cellular ontogeny, the adjectives ``differentiated'' or ``differentiating'' are relative terms. A "differentiated cell" can be a cell that is further along the developmental pathway than the cell being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into lineage-restricted cells (e.g., neuronal progenitor cells). can differentiate, and its progenitor cells can differentiate into terminally differentiated cells (e.g., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.) that play a characteristic role in a tissue type, and proliferate further. You may or may not be able to retain your abilities. Stem cells can be characterized by the presence of certain markers (eg, proteins, RNA, etc.) and the absence of certain markers. Stem cells can also be identified by both in vitro and in vivo functional assays, particularly those related to the ability of stem cells to give rise to a wide variety of differentiated progeny. In one embodiment, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, including pluripotent stem cells, and trophoblast stem cells.

[0135] Stem cells of interest, eg, stem cells that can be used in accordance with the present disclosure, can include pluripotent stem cells (PSCs). As used herein, the term "pluripotent stem cell" or "PSC" can refer to a stem cell capable of producing all cell types of an organism. Thus, PSCs can give rise to cells of all germ cell layers of an organism (e.g. endoderm, mesoderm, ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to the ectodermal, mesodermal, and endodermal tissues of the organism. Pluripotent stem cells of plants are capable of giving rise to all cell types of a plant (eg, roots, stems, leaves, and other cells).

[0136] The term "embryonic stem cell" (ESC) refers to a pluripotent stem cell isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC strains, such as hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)) are listed in the NIH Human Embryonic Stem Cell Registry. Stem cells of interest also include embryonic stem cells from other primates, such as rhesus stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, etc. (Thomson et al. (1998) ) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al. Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs can grow as flat colonies with a large nuclear-cytoplasmic ratio, clear borders, and prominent nucleoli. Furthermore, ESCs can express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for producing and characterizing ESCs can be found, for example, in US Pat. No. 7,029,913, US Pat. No. 5,843,780, and US Pat. Methods of expanding undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein in its entirety.

[0137] The term "embryonic germ stem cell" (EGSC) or "embryonic germ cell" or "EG cell" refers to a germ cell and / or a germ cell progenitor cell, e.g. a cell capable of becoming a primordial germ cell, e.g. a sperm and an egg. Refers to pluripotent stem cells derived from. Fetal embryonic cells (EG cells) are thought to have similar properties to the fetal stem cells described above. Examples of methods for producing and characterizing EG cells include, for example, U.S. Pat. No. 7,153,684; Matsui, Y. et al. (1992) Cell 70:841; (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al. (1996) Development, 122:1235.

[0138] The term "induced pluripotent stem cell" or "iPSC" refers to a pluripotent cell derived from a cell that is not a PSC (eg, a cell that is differentiated compared to a PSC). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and can grow as flat colonies with a large nuclear-cytoplasmic ratio, clear borders, and prominent nuclei. Additionally, iPSCs contain enzymes known to those skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. One or more important pluripotency markers can be expressed. Examples of methods for producing and characterizing iPSCs include, for example, U.S. Patent Publication Nos. 2009 / 0047263 and 2009 / 006874, each of which is incorporated herein in its entirety. 2, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875 and 2009 / 0304646. Generally, to produce iPSCs, somatic cells are injected with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram somatic cells to become pluripotent stem cells. provide. In some embodiments, dedifferentiated stem cells are induced and reprogrammed cells, such as, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced pluripotent stem cells or iPS cells. It may be.

[0139] In some embodiments, the stem cells used in accordance with the present disclosure are from a mammalian species, e.g., human, horse, bovine, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. You can get it. In some embodiments, a mixture of cells from suitable sources of endothelial, muscle, and / or neural stem cells is harvested from a mammalian donor for purposes of this disclosure. A preferred source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (eg, recruited), may be removed from the subject.

[0140] Other aspects of the disclosure provide isolated cells other than stem cells, and cells differentiated from any of the isolated stem cells described herein. Cells other than stem cells and isolated stem cells can be differentiated into any cell type. In some embodiments, the isolated stem cells or cells differentiated from non-stem cells described herein include fibroblasts, endothelial cells, definitive endoderm cells, gastrula cells, pancreatic endoderm cells, Pancreatic progenitor cells, pancreatic endocrine cells, islet cells (e.g. β cells, α cells, δ cells, or enterochromaffin (EC) cells), stem cell-derived β cells, stem cell-derived α cells, stem cell-derived δ cells, stem cell-derived enterochromatocytes fin (EC) cells, insulin-producing cells, insulin-positive β-like cells, hematopoietic stem cells, hematopoietic progenitor cells, muscle cells (e.g., cardiomyocytes, skeletal muscle cells, or smooth muscle cells), satellite stem cells, hepatocytes (e.g., liver or hepatic stellate cells), neuronal cells (eg, dopaminergic neurons), or immune cells (eg, T cells, B cells, macrophages, natural killer cells). Cells differentiated from isolated stem cells, or cells other than stem cells as described herein, have reduced immunogenicity compared to wild type cells of the same cell type.

[0141] In some embodiments, the cells described herein (eg, cells differentiated from isolated stem cells) are cells of the pancreatic lineage. In some embodiments, cells of the pancreatic lineage include definitive endoderm cells, gastrula cells, pancreatic endoderm cells, pancreatic progenitor cells, pancreatic endocrine cells, and pancreatic islet cells (e.g., β cells, AN cells, δ cells, enterochromaffin (EC) cells), and combinations thereof. Methods for differentiating stem cells into the pancreatic lineage are known in the art and are described, for example, in at least U.S. Patent Application Nos. 2015 / 0240212, 2015 / 0218522, 2022 / 0090020, and U.S. Patent No. 11,466,256. , International Publication No. 2022 / 147056, and International Publication No. 2022 / 192300, each of which is incorporated herein by reference.

[0142] In some embodiments, the cells described herein (eg, cells differentiated from isolated stem cells) are immune cells (eg, T cells, or natural killer cells). In some embodiments, the immune cells are further modified to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs). As used herein, "chimeric antigen receptor T cells (CART cells)" refer to T cells that have been genetically engineered to produce artificial T cell receptors for use in immunotherapy. "Chimeric antigen receptor (CAR)" as used herein refers to an immunoreceptor protein that has been genetically engineered to give T cells a new ability to target specific proteins. Chimeric antigen receptors are chimeric because they combine both antigen binding activity and T cell activity into a single receptor. "T cell receptor (TCR)" as used herein refers to a protein complex found on the surface of T cells or T lymphocytes. TCR is responsible for recognizing antigen fragments as peptides bound to MHC molecules. When the TCR binds to antigenic peptides bound to MHC molecules, T cells are activated through signal transduction, resulting in an adaptive immune response.

[0143] In some embodiments, cells differentiated from the isolated stem cells described herein have the same genetic modifications as the isolated stem cells from which they are differentiated, such as immunosuppressive factors (e.g., PDL1, CD47 , or HLA-G), and optionally one or more immunosuppressive factors (e.g., CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E). , HLA-G, C1 inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9), and / or Including decreased expression of MHC-I and MHC-II. In some embodiments, a cell described herein (e.g., a cell differentiated from an isolated stem cell) has an immunosuppressive factor (e.g., PDL1, CD47, or HLA-G) an increase of at least 100%, at least 2x, at least 5x, at least 10x, at least 50x, at least 100x or more). In some embodiments, increased expression of an immunosuppressive factor (eg, PDL1, CD47, or HLA-G) is induced or increased by interferon gamma. In some embodiments, cells (e.g., pancreatic islet cells or immune cells) differentiated from isolated cells (e.g., stem cells) described herein are compared to wild-type cells of the same cell type. , low immunogenicity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower).

[0144] In some aspects, this disclosure also contemplates isolated immune cells having any of the genetic modifications disclosed herein, compared to unmodified isolated immune cells of the same type. immunogenicity is reduced. Such isolated immune cells can be further modified to express a CAR or TCR.

[0145] Compositions and treatment methods As used herein, in some embodiments, a composition comprising any of the cells disclosed herein (e.g., a cell differentiated from any of the isolated stem cells disclosed herein) Further provided. In some embodiments, the composition comprises a population of pancreatic islet cells (eg, human pancreatic islet cells). In some embodiments, pancreatic islet cells are differentiated from any of the isolated stem cells disclosed herein.

[0146] In some embodiments, the population of pancreatic islet cells (eg, human pancreatic islet cells) described herein comprises NKX6.1-positive, ISL-positive cells and NKX6.1-negative, ISL-positive cells. In some embodiments, the population of islet cells (e.g., human islet cells) differentiated from the isolated stem cells described herein has more NKX6.1 than NKX6.1-negative, ISL-positive cells. positive, including ISL-positive cells. In some embodiments, the cells in the population are differentiated from any isolated stem cells described herein.

[0147] In some embodiments, the population of pancreatic islet cells (e.g., human pancreatic islet cells) differentiated from isolated stem cells described herein comprises NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1 positive cells, less than 12% of the cells in the population (e.g., about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3 %, about 2%, about 1%, or less) are NKX6.1-negative, ISL1-negative cells. In some embodiments, less than 10%, less than 8%, less than 6%, less than 4%, 1-11%, 2-10%, 2-12%, 4-12%, 6- 12%, 8-12%, 2-8%, 4-8%, 3-6%, or 3-5% are NKX6.1-negative and ISL1-negative cells. In some embodiments, 2-12%, 4-12%, 6-12%, 8-12%, 2-8%, 4-8%, 3-6% or 3-5 of the cells in the population. % are NKX6.1 negative and ISL1 negative cells. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0148] In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85% of the cells in the population ~95%, or about 90-95%, are ISL1 positive cells. In some embodiments, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85 of the cells in the population %, 60~80%, 60~75%, 60~70%, 65~90%, 65~85%, 65~80%, 65~75%, 65~70%, 70~90%, 70~85 %, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% are ISL1-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISL1 positive cells. . In some embodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 of the cells in the population %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% are ISL1 positive cells. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0149] In some embodiments, the population of pancreatic islet cells (eg, human pancreatic islet cells) described herein comprises more NKX6.1-negative, ISL1-positive cells than NKX6.1-positive, ISL1-positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1 negative, ISL1 positive cells. In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52 of the cells in the population %, 53%, 54%, or about 55% are NKX6.1 negative, ISL1 positive cells. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0150] In some embodiments, the population of islet cells described herein (e.g., human islet cells) includes cells that express insulin (e.g., cells that express insulin but not glucagon or somatostatin), glucagon express cells that express glucagon (eg, cells that express glucagon but not insulin or somatostatin), and cells that express somatostatin (eg, cells that express somatostatin but not insulin or glucagon). In some embodiments, expression of insulin in a cell of the composition indicates that the cell is an SC-β cell. In some instances, expression of glucagon and lack of expression of somatostatin in a cell of the composition suggests that the cell is an SC-α cell. In some embodiments, expression of somatostatin and lack of expression of glucagon in a cell of the composition indicates that the cell is an SC-δ cell. In some embodiments, the insulin-expressing cells are also glucose-responsive insulin-producing cells. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0151] In some embodiments, the population of islet cells described herein (e.g., human islet cells) comprises (a) 30-90%, 30-80%, 30-70% of the cells in the cell population; 30~60%, 30~50%, 30~40%, 40~90%, 40~80%, 40~70%, 40~60%, 40~50%, 50~90%, 50~80%, 50~70%, 50~60%, 60~90%, 60~80%, 60~7 0%, 70-90%, 70-80%, or 80-90% express insulin; (b) 5-40%, 5-35%, 5-30%, 5- 25%, 5~20%, 5~15%, 5~ 10%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 15~40%, 15~35%, 15~30%, 15~ 25%, 15~20%, 20~40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35% or 35-40% express glucagon , somatostatin is expressed and / or (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20%, 4 of the cells in the cell population. ~15%, 4~12%, 4~10%, 4~8%, 4~ 5%, 5~20%, 5~15%, 5~12%, 5~10%, 5~8%, 7~20%, 7~15%, 7~12%, 7~10%, 9~ 20%, 9~15%, 9~12%, 8~10%, 8~12%, 8~ 15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but not glucagon Including. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0152] In some embodiments, a population of islet cells (e.g., human islet cells) differentiated from isolated stem cells described herein comprises (a) 30-90% of the cells in the cell population, 30-90% of the cells in the cell population; 80%, 30~70%, 30 ~60%, 30~50%, 30~40%, 40~90%, 40~80%, 40~70%, 40~60%, 40~50%, 50~90%, 50~80%, 50 ~70%, 50~60%, 60~90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% express insulin; (b) 5-40%, 5-35% of the cells in the cell population; 5~30%, 5~25%, 5~20 %, 5~15%, 5~10%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 15~40%, 15~35 %, 15~30%, 15~25%, 15~20 %, 20~40%, 20~35%, 20~30%, 20~25%, 25~40%, 25~35%, 25~30%, 30~40%, 30~35% or 35~40 % express glucagon, but somato statins are not expressed; and (c) 3-20%, 3-15%, 3-12%, 3-10%, 3-8%, 3-5%, 4-20% of the cells in the cell population; 4~15%, 4~12%, 4~10%, 4~8%, 4~5%, 5~20%, 5~15%, 5~12%, 5~10%, 5~8%, 7~20%, 7~15%, 7~12%, 7~10%, 9~20%, 9~15%, 9~12%, 8~10%, 8~12%, 8 ~15%, 8-20%, 10-20%, 10-12%, 10-15%, 12-20%, 12-15% or 15-20% of cells express somatostatin but not glucagon Including not doing so. In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0153] In some embodiments, the percentage of cells expressing a marker provided herein is determined by flow cytometry. In some embodiments, the percentage of cells expressing a marker provided herein is determined by immunohistochemical analysis.

[0154] In some embodiments, the insulin-expressing cells (i.e., SC-β cells) in the population of pancreatic islet cells (e.g., human pancreatic islet cells) described herein exhibit glucose-stimulated insulin secretion (GSIS). . In some embodiments, the insulin-expressing cells (i.e., SC-β cells) in the population of pancreatic islet cells (e.g., human pancreatic islet cells) described herein exhibit glucose-stimulated insulin secretion (GSIS). Further maturation into cells (eg, further maturation in subjects after transplantation). In some embodiments, the cells in the population are differentiated from any of the isolated stem cells described herein.

[0155] In some embodiments, a composition comprising a cell described herein (e.g., a cell differentiated from an isolated stem cell described herein) (e.g., a pancreatic islet cell or an immune cell) A therapeutic composition. The therapeutic composition can further include a physiologically compatible solution, including, for example, artificial cerebrospinal fluid or phosphate buffered saline. Therapeutic compositions can be used to treat, prevent, or stabilize diseases (eg, diabetes or cancer).

[0156] In some embodiments, the therapeutic composition includes other activating agents, such as anti-inflammatory agents, exogenous small molecule agonists, exogenous small molecule antagonists, anti-apoptotic agents, antioxidants, and / or It further contains growth factors known in the art.

[0157] In some embodiments, the therapeutic composition further comprises a pharmaceutically acceptable carrier (eg, a medium or excipient). The term pharmaceutically acceptable carrier (or vehicle) can be used interchangeably with the term biologically compatible carrier or vehicle, and is compatible with the cells and other agents being therapeutically administered. Reagents, cells, compounds, and materials that are not only suitable for use in contact with human and animal tissues, but also without undue toxicity, irritation, allergic responses, or other complications. , composition, and / or dosage form. Suitable pharmaceutically acceptable carriers include water, saline (eg, Ringer's solution), alcohol, oil, gelatin, and carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone. I can do that. Such preparations are sterile and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, and colorants. can be done. Pharmaceutical compositions containing components or products of cells other than living cells can be formulated as liquids. Pharmaceutical compositions containing living non-native pancreatic β cells can be formulated as liquids, semisolids (eg, gels, gel capsules, or liposomes), or solids (eg, matrices, scaffolds, etc.).

[0158] In some embodiments, the therapeutic composition comprises one or more physiologically acceptable excipients and adjuvants that facilitate processing of the active compound into a preparation that can be used pharmaceutically. It is formulated in conventional manner using carriers. Proper formulation is dependent upon the route of administration chosen. A summary of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel. Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition (Lippincott Williams & Wilkins 1999).

[0159] In some embodiments, the therapeutic composition is optionally prepared in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, dragaging, suspending, emulsifying, encapsulating, Manufactured by encapsulation or compression processes.

[0160] In some embodiments, the therapeutic composition comprises acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid and hydrochloric acid; sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate; bases such as sodium lactate and tris-hydroxymethylaminomethane; and one or more pH adjusting agents or buffers, including buffers such as citric acid / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0161] In some embodiments, the therapeutic composition further comprises one or more salts in an amount necessary to provide an acceptable osmotic pressure of the composition. Such salts include salts with sodium, potassium or ammonium cations and chloride, citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfate, thiosulfate or bisulfite anions; preferred salts include: Mention may be made of sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfate and ammonium sulfate.

[0162] In some embodiments, therapeutic compositions can be administered orally, without limitation, parenterally (e.g., intravenously, subcutaneously, intramuscularly, intracerebral, intraventricularly, intraarticularly, intraperitoneally, or intracranially), intranasally. Suitable for administration by any route of administration, including intrabuccal, sublingual, or rectal routes of administration. In some embodiments, therapeutic compositions are formulated for parenteral (eg, intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, or intracranial) administration.

[0163] In some embodiments, the therapeutic composition further comprises one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride. It will be done.

[0164] In some embodiments, the therapeutic composition comprises an amount of a population of pancreatic islet cells described herein (e.g., a monomer described herein) effective to treat or prevent diabetes, e.g. (a population of pancreatic islet cells differentiated from either isolated stem cells). In some embodiments, the therapeutic composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffering agents, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as Glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.

[0165] In some embodiments, therapeutic compositions comprising cells, cell components, or cell products are delivered to the patient's kidneys in one or more of several delivery methods known in the art. I can do that. In some embodiments, the composition is delivered to the kidney (eg, above and / or below the renal capsule). In another embodiment, the composition may be delivered to various locations within the kidney by periodic intraperitoneal or intrarenal injections. Alternatively, the compositions may be applied in other dosage forms known to those skilled in the art, such as preformed or in situ formed gels or liposomes.

[0166] In some embodiments, therapeutic compositions comprising living cells in a semi-solid or solid carrier can be formulated for surgical implantation onto or under the renal capsule. It should be appreciated that liquid compositions may also be administered by surgical means. In certain instances, semi-solid or solid pharmaceutical compositions can include semipermeable gels, lattices, cell scaffolds, etc., which may be non-biodegradable or biodegradable. For example, in certain cases, it is possible to isolate an exogenous cell from its surroundings and also allow the cell to secrete and deliver biological molecules (e.g., insulin) to surrounding cells or the bloodstream. , may be desirable or appropriate. In these cases, the cells can be formed as autonomous grafts containing living cells with a non-degradable, selectively permeable barrier that physically separates the transplanted cells from the host tissue. Such grafts are sometimes referred to as "immunoprotective" due to their ability to prevent immune cells and macromolecules from killing the transplanted cells in the absence of pharmacologically induced immunosuppression. A variety of encapsulation devices, degradable gels and networks can be used in the pharmaceutical compositions of the present disclosure. For example, degradable materials particularly suitable for sustained release formulations include biocompatible polymers such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.

[0167] In some embodiments, it may be desirable or appropriate to deliver cells on or into a biodegradable, preferably bioresorbable or bioabsorbable scaffold or matrix. These typical three-dimensional biomaterials contain living cells embedded in an extracellular matrix attached to, dispersed within, or entrapped in a scaffold. Once implanted in the target area of ​​the body, these grafts become integrated with the host tissue and the transplanted cells become established over time. Examples of scaffold or matrix (sometimes collectively referred to as "frameworks") materials that can be used in this disclosure include nonwoven mats, porous foams, or self-assembled peptides. Nonwoven mats can be made of fibers containing, for example, synthetic absorbent copolymers of glycolic acid and lactic acid (PGA / PLA), foams, and / or poly(epsilon-caprolactone) / poly(glycolic acid) (PCL / PGA) copolymers. It may be formed using

[0168] In some embodiments, the framework is a felt, which may be composed of multi-threads made from bioabsorbable materials, such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid. This yarn is felted using standard textile processing techniques consisting of crimping, cutting, carding and needling. In another embodiment, cells are seeded onto a foam scaffold, which can be a hybrid structure. In many of the embodiments described above, the framework can be molded into any useful shape. Additionally, non-native pancreatic beta cells can be cultured on preformed, non-degradable surgical or implantable devices.

[0169] In some embodiments, the matrix, scaffold or device can be treated prior to cell seeding to enhance cell attachment. For example, prior to inoculation, a nylon matrix can be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and / or collagen to coat the nylon. Polystyrene can be similarly treated using sulfuric acid. The external surface of the framework may also be coated with, for example, a plasma coating of the framework or one or more proteins (e.g., collagen, elastic fibers, reticular fibers) to improve cell adhesion or growth and tissue differentiation. Glycoproteins, glycosaminoglycans (e.g. heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), cellular matrices, and / or gelatin, alginic acid, among others, but not limited to Modifications can be made, such as by the addition of other materials such as salt, agar, agarose, and vegetable gums.

[0170] In some aspects, the present disclosure provides a device comprising a population of pancreatic islet cells described herein (e.g., a population of pancreatic islet cells differentiated from any of the isolated stem cells described herein). Provide. In some embodiments, the islet cells form cell clusters. The device can be configured to house cells described herein that, in certain embodiments, produce and release insulin when implanted into a subject. In some embodiments, the device can further include a semipermeable membrane. The semipermeable membrane can be configured to retain the cell clusters within the device and allow passage of insulin secreted by the cells. In some examples of devices, cells can be encapsulated by a semipermeable membrane. Encapsulation may be performed by any technique available to those skilled in the art. Semipermeable membranes can also be made from any suitable material, as those skilled in the art will recognize and appreciate. For example, semipermeable membranes can be made from polysaccharides or polycations. In some examples, semipermeable membranes include poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other polyhydroxy acids, poly(caprolactone). ), polycarbonates, polyamides, polyanhydrides, polyphosphazenes, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, biodegradable polyurethanes, albumin, collagen, fibrin, polyamino acids, prolamines, alginates, agaroses, including gelatin , dextran, polyacrylates, ethylene-vinyl acetate polymers and other acyl-substituted cellulose acetates and their derivatives, polyurethanes, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, or It can be made from any combination thereof. In some examples, the semipermeable membrane includes alginate. In some embodiments, cells are encapsulated in microcapsules that include an alginate core surrounded by a semipermeable membrane. In some embodiments, the alginate core is modified to produce a scaffold that includes an alginate core having an oligopeptide covalently conjugated with, for example, an RGD sequence (arginine, glycine, aspartate). In some instances, the alginate core is modified to produce covalently reinforced microcapsules, eg, having chemoenzymatically engineered alginates with increased stability. In some embodiments, the alginate core is modified to produce a membrane-mimetic film assembled by, for example, in-situ polymerization of acrylate-functionalized phospholipids. In some examples, microcapsules are constructed from alginate that has been enzymatically modified using epimerase. In some instances, the microcapsules include covalent bonds between adjacent layers of the microcapsule membrane. In some embodiments, the microcapsules include subsieve capsules that include alginate linked with a phenolic moiety. In some examples, the microcapsules include a scaffold that includes alginate-agarose. In some embodiments, the cells are modified with PEG before being encapsulated within the alginate. In some embodiments, cells are encapsulated within photoreactive liposomes and alginate. Alginates used in the microcapsules include, but are not limited to, polyethylene glycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronic acid, dextran including ROD, BHD and polyethylene glycol-diacrylate (PEGDA), poly(MPC- other suitable biomaterials, including co-n-butyl methacrylate-co-4-vinylphenylboronic acid (PMBV) and poly(vinyl alcohol) (PVA), agarose, agarose, including gelatin, and multilayered examples thereof. can be replaced with In some embodiments, the devices provided herein include an extracorporeal segment, eg, a portion of the device may reside outside the subject's body when the device is implanted in the subject. The extracorporeal segment may include any functional component of the device, whether or not including cells or cell clusters provided herein.

[0171] Further provided herein are methods for treating or preventing disease in a subject. Compositions comprising pancreatic islet cells differentiated from isolated stem cells described herein can be administered to a subject to restore a degree of pancreatic function to the subject. In some embodiments, such compositions are implanted into a subject. The term "transplantation" refers to a cell or cell cluster, a cell or any part of a cell cluster, or a cell, by a method or route that results in at least partial localization of the introduced cell or cell cluster at the desired site. It can refer to the placement into a subject of any composition comprising cell clusters or any portion thereof. In some embodiments, the desired site is the pancreas. In some embodiments, the desired site is a location other than the pancreas, e.g., in the liver or in a capsule (e.g., a microcapsule) to maintain the transplanted cells at the transplanted location and avoid migration. It is subcutaneous. In some embodiments, the transplanted cells release insulin in an amount sufficient to lower the subject's blood glucose level.

[0172] In some embodiments, a composition comprising a pancreatic islet cell disclosed herein (e.g., a pancreatic islet cell differentiated from any of the isolated stem cells described herein) is transplanted into a subject. It is housed in a device. In some embodiments, a device implanted in a subject releases insulin while retaining cells within the device and promotes tissue angiogenesis within and around the device. Exemplary devices include, for example, International Application Publication No. WO2018 / 232180, International Application Publication No. WO2019 / 068059, International Application Publication No. WO2019 / 178134, International Application Publication No. WO2020 / 206150, and International Application Publication No. WO2020 No. 206157, each of which is incorporated by reference in its entirety. In some embodiments, the subject is not administered an immunosuppressant during device implantation or angiogenesis. In some embodiments, the device has a thickness of at least about 300 pm. In some embodiments, the device comprises a membrane that includes multiple nodes interconnected by multiple fibrils.

[0173] In some embodiments, the device includes a first membrane having a first surface including a plurality of channels and a plurality of second surfaces opposite the first surface; a second membrane attached opposite the second surface, the first membrane and the second membrane forming a sealed compartment having a surface area to volume ratio of at least about 40 cm-l; Provide a volume for housing cells within the device.

[0174] In some embodiments, the enclosed compartment comprises a single continuous open chamber. In some embodiments, the volume is about 8 μL to about 1,000 μL. In some embodiments, the device has a length and / or width of about 0.25 cm to about 3 cm. In some embodiments, the device has a thickness of at least about 300 pm.

[0175] In some embodiments, the plurality of channels are generally perpendicular to the first membrane. In some embodiments, the plurality of channels are arranged in a straight line. In some embodiments, the plurality of channels are arranged in a polar array. In some embodiments, the channels have an average diameter of about 400 pm to about 3,000 pm. In some embodiments, the diameter is measured at the narrowest part of the channel. In some embodiments, the center of each channel is separated from the center of another channel by a distance of about 75 pm to about 500 pm. In some embodiments, the channel has a height to diameter ratio of at least about 0.2. In some embodiments, the device has a number of channels per area along the cross section, and in some examples the number is greater than about 50 / cm2.

[0176] In some embodiments, at least one of the first membrane and the second membrane includes a plurality of nodes interconnected by a plurality of fibrils. In some embodiments, at least one of the first membrane and the second membrane comprises PVDF, PTFE, ePTFE, PCL, PE / PES, PP, PS, PMMA, PLGA, PLLA, or any combination thereof. include. In some embodiments, the device further comprises an opening through the first membrane and / or the second membrane within the channel. In some embodiments, the opening has a concentricity with the channel of at most about 25% of the diameter of the channel. In some embodiments, it is a frame configured to receive a device described herein. In some embodiments, the frame is configured to receive multiple cell containment devices. In some embodiments, the frame includes a bending mechanism configured to prevent buckling of the cell containment device.

[0177] In some embodiments, the methods described herein produce a pancreatic islet cell as described herein (e.g., a pancreatic islet cell differentiated from any of the isolated stem cells described herein). into the subject using any means in the art. For example, the method may include implanting the cell cluster intraperitoneally, through the portal vein, the subrenal space, the renal capsule, the theca, the subcutaneous space, or via pancreatic bed injection. For example, the implantation can be subcapsular, intramuscular, or intraportal, eg, intraportal injection. Immunoprotective encapsulation can be performed to render the cell cluster immunoprotective. In some examples, the methods of treatment provided herein include one or more immune responses to modulate or reduce a transplant rejection response or other immune response to a transplant (e.g., a cell or device). may include administering a response modulator. Examples of immune response modulators that can be used in this method include purine synthesis inhibitors such as azathioprine and mycophenolic acid, pyrimidine synthesis inhibitors such as leflunomide and teriflunomide, antifolates such as methotrexate, tacrolimus, Cyclosporine, pimecrolimus, avetimus, gusperilimus, lenalidomide, pomalidomide, thalidomide, PDE4 inhibitors, apremilast, anakinra, sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, anti-thymocyte globulin antibody, anti-lymphocyte globulin antibody, CTLA- 4, fragments thereof and fusion proteins thereof such as abatacept and belatacept, TNF inhibitors such as etanercept and pegsnercept, antibodies against complement component 5 such as aflibercept, alefacept, rilonacept, eculizumab, adalimumab, afelimomab, Sertoli Anti-TNF antibodies like zumab pegol, golimumab, infliximab, and nerelimomab, antibodies against interleukin-5 like mepolizumab, anti-IgE antibodies like omalizumab, anti-interferon antibodies like fararimomab, anti-IL like ercilimomab -6 antibodies, antibodies against IL-12 and IL-23 like lebrikizumab and ustekinumab, anti-IL-17 antibodies like secukinumab, muromonab-CD3, anti-CD3 antibodies like otelixizumab, teplizumab, and bicilizumab, crenoliximab, keliximab , and anti-CD4 antibodies like zanolimumab, anti-CD11a antibodies like efalizumab, anti-CD18 antibodies like erulizumab, anti-CD20 antibodies like obinutuzumab, rituximab, ocrelizumab and pascolizumab, anti-CD23 antibodies like gomiliximab and lumiliximab, Anti-CD40 antibodies such as teneliximab and tralizumab, antibodies against CD62L / L-selectin such as acerizumab, anti-CD80 antibodies such as galiximab, anti-CD147 / basidin antibodies such as gavilimomab, anti-CD154 antibodies such as luplizumab, belimumab and Anti-BLyS antibodies such as brisibimod, anti-CTLA-4 antibodies such as ipilimumab and tremelimumab, anti-CAT antibodies such as Bertilimumab, lerdelimumab, and metelimumab, anti-integrin antibodies such as natalizumab, and interleukins such as tocilizumab. Antibodies against the -6 receptor, anti-LFA-1 antibodies such as odulimomab, basiliximab, dacilizumab, and antibodies against the IL-2 receptor / CD25 such as inolimomab, atrolimumab, cedelizumab, fontolizumab, maslimomab, morolimumab , pexelizumab, reslizumab, lobelizumab, ciplizumab, talizumab, telimomab alitox, vapariximab, and bepalimomab, such as antibodies against T lymphocytes (zolimomab alitox).

[0178] As used herein, the terms "treating" and "treatment" mean that the subject is suffering from a reduction in at least one symptom of a disease or an amelioration of the disease, e.g., a beneficial or desired effect. It can refer to administering to a subject an effective amount of a composition (eg, a cell cluster or a portion thereof) to have a clinical result. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, attenuation of the severity of a disease, whether detectable or undetectable, stabilization (eg, no worsening), slowing or slowing of disease progression, amelioration or alleviation of disease status, and remission (eg, partial or total). Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, those skilled in the art will appreciate that treatment may ameliorate the disease condition, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prophylaxis.

[0179] Exemplary modes of administration include, but are not limited to, injection, infusion, eye drops, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intrasaccular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, and joint. These include intrathecal, subcapsular, intrathecal, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In a preferred embodiment, the composition is administered by intravenous injection or infusion.

[0180] “Treatment,” “prevention,” or “amelioration” of a disease or disorder means delaying the onset or preventing the onset of, or relating to, such a disease or disorder. It means to reverse, alleviate, ameliorate, inhibit, slow or stop the progression, severity or aggravation, progression or severity of a condition. In one embodiment, one or more symptoms of the disease or disorder are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to untreated subjects. eased.

[0181] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to lower sugar levels to near normal as safely as possible. Commonly set goals are 80-120 milligrams per deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different targets for a patient depending on other factors, such as how much of a hypoglycemic response the patient often has. Useful medical tests include tests on a patient's blood and urine to determine blood sugar levels, glycosylated hemoglobin level (HbA1c; measurement of average blood glucose level over the past 2-3 months, normal range is 4-6 ), cholesterol and fat levels, and urine protein levels. Such tests are standard tests known to those skilled in the art (see, eg, American Diabetes Association, 1998). An effective treatment program can also be determined by the fact that in the program the patient has fewer complications related to diabetes, such as eye disease, kidney disease, or neurological disease.

[0182] Delaying the onset of diabetes in a subject by at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years , at least one symptom of diabetes, such as hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, for a period of at least 30 years, at least 40 years, or longer, which may include the subject's entire lifespan. blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neurosis, autonomic dysfunction, hyperglycemic hyperosmolar coma, or Refers to the delay in the start of these combinations.

[0183] In some embodiments, the reduction in a subject's blood glucose level induced by implantation of a cell, composition or device provided herein results in an amount of glucose below the diabetic threshold. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human. In some embodiments, the amount of glucose is lowered below the diabetes threshold 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after transplantation.

[0184] Subjects that can be treated by the methods herein can be human or non-human animals. In some examples, the subject can be a mammal. Examples of subjects include, but are not limited to, primates, such as monkeys, chimpanzees, bamboos, or humans. In some examples, the subject is a human. The subject may be a non-primate animal including, but not limited to, dogs, cats, horses, cows, pigs, sheep, goats, rabbits, and the like. In some instances, the subject undergoing treatment is a subject in need thereof, such as a human in need thereof.

[0185] The terms "patient" and "subject" are used interchangeably herein. Preferably the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Mammals other than humans can be advantageously used as subjects to represent animal models of type 1 diabetes, type 2 diabetes mellitus, or prediabetes. Additionally, the methods described herein can be used to treat livestock and / or pets. The subject may be male or female. The subject has or has been previously diagnosed or identified as having diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or prediabetes, and optionally and may have never had to undergo treatment for diabetes, one or more complications associated with diabetes, or prediabetes. The subject may not have diabetes or prediabetes. The subject has been diagnosed or identified as having diabetes, one or more complications associated with diabetes, or prediabetes, but has diabetes, one or more complications associated with diabetes, or diabetes. The person may also exhibit improvement in known diabetes risk factors as a result of receiving one or more treatments for the pre-existing condition. Alternatively, the subject may be one who has not been previously diagnosed with diabetes, one or more complications associated with diabetes, or prediabetes. For example, a subject may be one who exhibits one or more risk factors for diabetes, diabetes-related complications, or prediabetes, or a subject who does not exhibit diabetes risk factors, or one or more diabetes-related The subject may be asymptomatic for complications of diabetes or prediabetes. The subject may have diabetes or prediabetes or be at risk of developing diabetes or prediabetes. The subject may be one who has been diagnosed or identified as having one or more complications associated with diabetes or prediabetes, as defined herein, or the subject may have diabetes, as defined herein. The person may not have been previously diagnosed or identified as having one or more complications associated with or prediabetes.

[0186] In some aspects, the present disclosure provides an immune cell described herein (e.g., an immune cell differentiated from an isolated stem cell described herein), or a gene described herein. Provided are methods of treating cancer by administering to a subject immune cells that include modifications or are genetically modified using the methods described herein, or compositions that include such immune cells. do. In some embodiments, the immune cells further express chimeric antigen receptors or engineered T cell receptors. In certain embodiments, the subject is a mammal, such as a primate, such as a human. Non-limiting examples of cancers that may be treated according to the present disclosure include adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancers, anal cancer, appendiceal cancer, Astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant nerve Glioma, glioblastoma, ependymoma, medulloblastoma, supraventricular primitive neuroectodermal tumor, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, cancer of unknown primary origin, Central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia and myeloid leukemia, chronic myeloproliferative diseases, colon cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumor, extracranial germ cell tumor, extramandibular germ cell tumor, extrahepatic cholangiocarcinoma, intraocular melanoma, retinoblast. cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, extracranial germ cell tumor, extramaxillary germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, Hepatocellular carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual tract glioma, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer. , lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenström giant cell tumor, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, Malignant mesothelioma, squamous cell neck cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disease, chronic myeloproliferative disease, nasal cavity / sinus cancer , nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and antrum. Primitive neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous cell neck cancer, supraventricular primary neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, Transitional cell carcinoma, choriocarcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematological neoplasm, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumor and germ cell tumors, or Wilms tumor. In some embodiments, the cancer is metastatic cancer.

[0187] In some aspects, the present disclosure relates to cardiomyocytes, skeletal muscle cells, smooth muscle cells, and / or satellite stem cells (e.g., myocytes or satellite stem cells differentiated from isolated stem cells described herein). Myopathies (e.g., Duchenne muscular dystrophy or myotonic dystrophy) by administering to a subject any of the myocytes described herein, such as, or a composition comprising such myocytes or satellite stem cells. provide a method for treating

[0188] In some aspects, the present disclosure provides for administering to a subject a hematopoietic progenitor cell (e.g., a hematopoietic progenitor cell differentiated from any of the stem cells disclosed herein), or a composition comprising such a hematopoietic progenitor cell. Provided are methods of treating blood diseases (eg, beta thalassemia or sickle cell disease) by.

[0189] In some aspects, the present disclosure provides hepatocytes, such as hepatocytes or hepatic stellate cells (e.g., hepatocytes differentiated from any of the stem cells disclosed herein), or comprising such hepatocytes. Methods of treating liver disorders (eg, alpha-1 antitrypsin deficiency) are provided by administering compositions to a subject.

[0190] In some aspects, the present disclosure provides neuronal cells, such as dopaminergic neurons (e.g., dopaminergic neurons differentiated from any of the stem cells disclosed herein), or compositions comprising such neuronal cells. A method of treating a neurological disease (eg, Parkinson's disease) by administering an agent to a subject is provided. How to produce hypoimmune cells In some aspects, the disclosure provides methods of producing isolated cells (eg, isolated stem cells) described herein. In some embodiments, the method of producing an isolated cell comprises modifying the genome of the cell to introduce a genetic modification described herein (e.g., an allele encoding an immunosuppressive factor). inserting an exogenous polynucleotide sequence encoding one or more immunosuppressive factors, inserting an exogenous polynucleotide sequence encoding an anti-CRISPR protein, and / or (disrupting genes that reduce the expression of MCH-I or MHC-II). The genetic modifications described herein can be performed by any method available to those skilled in the art.

[0191] In some embodiments, the methods of producing cells (e.g., isolated stem cells) described herein include using a gene editing system capable of making the general purpose modifications described herein. (eg, human embryonic stem cells, human pluripotent stem cells, or human induced pluripotent stem cells). For example, in some embodiments, the gene editing system is a CRISPR-Cas gene editing system. Such systems include, for example, one or more endonucleases and one or more guide RNAs that target the gene sequence of interest. In some embodiments, the hypoimmune cells described herein are obtained by introducing a CRISPR-Cas gene editing system into the stem cells to generate one or more immunosuppressive genes in the 3'-UTR of one or more immunosuppressive genes. Produced by causing destruction.

[0192] In some embodiments, the present disclosure provides one or more guide RNAs (gRNAs) comprising a nucleotide sequence that targets the 3'-UTR of an allele encoding an immunosuppressive factor, or one encoding a gRNA. Alternatively, a method is provided in which a hypoimmune cell is produced by delivering a composition comprising a plurality of nucleic acids to a cell (eg, a stem cell). In some embodiments, the gene editing system includes a nuclease (eg, an endonuclease) or a recombinase (eg, a site-specific recombinase) that can destroy the target genomic site. Non-limiting examples of nucleases (e.g., endonucleases) that can be used in accordance with the present disclosure include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and RNA-guided endonucleases (e.g. , CRISPR-Cas9 or CRISPR / Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9 nuclease). Non-limiting examples of recombinases (e.g., site-specific recombinases) that may be used in accordance with the present disclosure include Cre, Bxb1, FLPe, phiC31 integrase, phiC31 exosigenase, R4, PhiBT1, Wβ integrase, SPBc, and TP901- 1 is mentioned. In some embodiments, the gene editing system comprises a nucleic acid encoding a nuclease (eg, endonuclease) or recombinase (eg, site-specific recombinase) that can destroy the target genomic site. In some embodiments, the gene editing system includes a transposon system, such as the piggyBac transposon system.

[0193] In some embodiments, the gene editing system comprises a zinc finger nuclease (ZFN) or a nucleic acid encoding a ZFN. Zinc finger nucleases (ZFNs) are targeted nucleases that contain a nuclease fused to a zinc finger DNA binding domain (ZFBD), a polypeptide domain that binds to DNA in a sequence-specific manner through one or more zinc fingers. . Zinc fingers are approximately 30 amino acid domains within the zinc finger binding domain, the structure of which is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain that does not exist in nature, and whose design / configuration is based primarily on rational criteria, e.g. processing information in a database storing information of existing ZFP designs and combined data. results from the application of substitution rules and computerized algorithms to See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; and WO 98 / 53058; and 03 / 016496. The zinc finger domains selected are domains not found in nature, produced primarily from empirical processes such as phage display, interaction traps or hybrid selection. ZFNs are further described in US Pat. No. 7,888,121 and US Pat. No. 7,972,85. The best recognized example of a ZFN is a fusion of the FokI nuclease and a zinc finger DNA binding domain.

[0194] In some embodiments, the gene editing system comprises a transcription activator-like effector nuclease (TALEN) or a nucleic acid encoding a TALEN. Transcription activator-like effector nucleases (TALENs) are targeted nucleases that include a nuclease fused to a transcription activator-like effector DNA binding domain. A "transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" is a polypeptide domain of a TAL effector protein that is responsible for binding the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences through their DNA-binding domains, and activate gene transcription at these sequences through their transactivation domains. The specificity of the TAL effector DNA-binding domain relies on a variable number of imperfect 34 amino acid repeats of the effector, which contain polymorphisms at selective repeat positions termed repeat variable diresidues (RVDs). TALENs are described in more detail in US Patent Application Publication No. 2011 / 0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of FokI nuclease and a TAL effector DNA binding domain.

[0195] In some embodiments, the gene editing system comprises an RNA guided nuclease or a nucleic acid encoding an RNA guided nuclease. RNA-guided endonucleases are enzymes that utilize RNA:DNA base pairing to target and cleave polynucleotides. The RNA-guided endonuclease can cleave at least one strand of a single-stranded polynucleotide or a double-stranded polynucleotide. The gene editing system may include one RNA-guided endonuclease. Alternatively, the gene editing system comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) RNA-guided endonucleases. may include. In some embodiments, the gene editing system encodes one or more (e.g., 1, 2, 3, 4, 5, or more) guide RNAs (gRNAs), or one or more gRNAs. further comprising one or more nucleic acids. In some embodiments, the gene editing system includes a nucleotide acid encoding both an RNA-guided nuclease and one or more gRNAs. In some embodiments, the gene editing system includes one or more (eg, 1, 2, 3, 4, 5, or more) RNA-guided nucleases and a gRNA complex.

[0196] In some embodiments, the gene editing system comprises a CRISPR / Cas system and the RNA guided nuclease is a Cas protein. In some embodiments, the Cas protein comprises a Cas core protein. Exemplary Cas core proteins include Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas12i, Cas14, Casφ, and modified versions thereof. but not limited to. Cas proteins and variants that can be used in gene editing are well known in the art, as described, for example, in Xu et al. known in the art, the entire contents of each of which are incorporated herein by reference.

[0197] In some embodiments, the Cas protein comprises an E. coli subtype Cas protein (also known as CASS2). Exemplary Cas proteins of the E. coli subtype include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Dvulg subtype (also known as CASS1) Cas protein. Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Tneap subtype (also known as CASS7) Cas protein. Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments, the Cas protein comprises a Hmari subtype Cas protein. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0198] In some embodiments, the Cas protein is the Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is the Staphylococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is the Streptococcus thermophilus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is the Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is the Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein or a functional portion thereof from any bacterial species. Cas9 protein is a member of the type II CRISPR system, which typically includes transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas proteins. An example of the Cas9 protein from Streptococcus pyogenes is a polypeptide containing 1368 amino acids (described in Uniprot accession number Q99ZW2). Cas9 has a RuvC-like domain (residues 7–22, 759–766, and 982–989) that cleaves target DNA that is not complementary to crRNA, and an HNH nuclease domain (residues 810 and 989) that cleaves target DNA that is complementary to crRNA. Contains two endonuclease domains, including ~872). In some embodiments, one or both of the HNH or RuvC-like domains are non-functional.

[0199] In some embodiments, the Cas protein is capable of generating double-strand breaks, e.g., as described in Komor et al., Nature, May 19, 2016;533(7603):420-4. It contains a catalytically inactive Cas (eg, Cas9) domain fused to an enzyme (eg, adenosine deaminase) that can be mutated without any modification.

[0200] In some embodiments, the Cas protein comprises a catalytically inactive Cas (eg, Cas9) domain fused to a reverse transcriptase. In some embodiments, such a Cas protein specifies the target site and encodes the desired edit, e.g., as described in Anzalone et al., Nature 576:149-157 (2019). can be used with prime editing guide RNA (pegRNA) to

[0201] In some embodiments, the Cas protein is a Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 or a functional portion thereof from any bacterial species. In some embodiments, Cpf1 is Francisella novicida U112 protein or a functional portion thereof. In some embodiments, Cpf1 is Acidaminococcus BV3L6 protein or a functional portion thereof. In some embodiments, Cpf1 is the Lachnospiraceae bacterial ND2006 protein or a functional portion thereof. The Cpf1 protein is a member of the type V CRISPR system. Cpf1 protein is a polypeptide containing approximately 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Cpf1 uses a single ribonuclease domain to cut target DNA in a staggered manner. Staggered DNA double-strand breaks result in 4 or 5-nt 5' overhangs.

[0202] In some embodiments, the Cas protein is a Cas12i protein, or a functional portion thereof, as described in WO2019178427. In some embodiments, the Cas12i protein is a type VI (CLUST.029130) Cas protein. In some embodiments, the Cas12i protein is about 1100 amino acids or less in length (and includes at least one RuvC domain).

[0203] In some embodiments, the Cas protein is a CasPhi or Cas14 protein. As used herein, "functional moiety" refers to a portion of a peptide that retains the ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence. . In some embodiments, the functional moiety comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. include. In some embodiments, the functional moiety comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. include. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain. In some embodiments, the functional portion of the Cpf1 protein comprises a functional portion of the RuvC-like domain.

[0204] The RNA-guided nucleases (eg, Cas proteins) described herein are guided to a target genomic site by one or more gRNAs. Naturally, two non-coding RNAs-crisprRNA (crRNA) and transactivating RNA (tracrRNA) direct mRNA-guided nucleases (eg, Cas proteins) to target genomic sites. crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through typical Watson-Crick base pairing with a 20 nucleotide (nt) sequence in the target DNA. By changing the 5'20nt base sequence of crRNA, the CRISPR-Cas9 complex can be targeted to specific genetic loci. The CRISPR-Cas9 complex will only bind to DNA sequences containing sequences matching the first 20 nt of crRNA if the target sequence is followed by a specific short DNA motif called the protospacer-adjacent motif (PAM). TracrRNA hybridizes with the 3' end of crRNA to form an RNA duplex structure and binds with Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which then cleaves the target DNA. I can do it. When the CRISPR-Cas9 complex binds to the DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, resulting in base-paired termination of both strands of DNA. (Blunt end) Double strand break (DSB) remains.

[0205] In some embodiments, the gRNA is a dual guide RNA or a single guide RNA (sgRNA). In some embodiments, the guide RNA is a single guide RNA (sgRNA) that includes aspects of both tracrRNA and crRNA.

[0206] In some embodiments, the gene editing system used in the methods of producing isolated cells (e.g., isolated stem cells) described herein comprises one or more gRNAs or one or comprises one or more nucleic acids encoding multiple gRNAs. gRNAs can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR / Cas system employed and the sequence of the target polynucleotide, as will be understood by those skilled in the art. obtain.

[0207] As will be understood by those skilled in the art, each gRNA is designed to include a spacer sequence that is complementary to its genomic target sequence. See Jinek et al., Science, 337, pp. 816-821 (2012) and Deltcheva et al., Nature, 471, pp. 602-607 (2011). A spacer sequence is a sequence (eg, a 20 base sequence) that defines a target sequence of a target nucleic acid of interest (eg, a DNA target sequence such as a genomic target sequence). The gRNA can include a variable length spacer sequence having 17-30 nucleotides at the 5' end of the gRNA sequence. In some embodiments, the spacer sequence is 15-30 nucleotides. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence is 20 nucleotides.

[0208] A "target sequence" is a sequence that flanks the PAM sequence and is modified by an RNA-guided nuclease (eg, Cas9). A "target nucleic acid" is a double-stranded molecule, one strand containing the target sequence and referred to as the "PAM strand" and the other complementary strand referred to as the "non-PAM strand." Those skilled in the art will recognize that gRNA spacer sequences hybridize to the reverse complement of the target sequence located on the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. The gRNA spacer interacts with the target nucleic acid in a sequence-specific manner through hybridization (ie, base pairing). Therefore, the base sequence of the spacer varies depending on the target sequence of the target nucleic acid of interest.

[0209] The spacer sequence is designed to hybridize to a region of the target nucleic acid located 5' to the PAM of the Cas9 enzyme used in the system. A spacer may be a perfect match to the target sequence, or it may be a mismatch. Each Cas protein has a specific protein that it recognizes in the target DNA, as described, for example, in Xu et al., Computational and Structural Biotechnology Journal, vol. It has a PAM sequence. For example, S. pyogenes Cas9 recognizes a PAM containing the sequence 5'-NRG-3' in a target nucleic acid, where R includes either A or G and N is any nucleotide. and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence. In another example, the Cas12i protein contains a PAM containing the sequence 5'-TTN-3' or 5'-TTH-3' or 5'-TTY-3' or 5'-TTC-3' in the target nucleic acid. where N is any nucleotide, H is adenine, cytosine, or thymine, and Y is cytosine, thymine, or pyrimidine.

[0210] In some embodiments, the target nucleic acid sequence comprises 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20 bases immediately 5' of the first nucleotide of the PAM. For example, 5’-NNNNNNNNNNNNNN N.R.G. -3'-containing sequences, the target nucleic acid includes a sequence corresponding to Ns, where N is any nucleotide, and the underlined NRG sequence is S. aureus PAM.

[0211] In some embodiments, the gRNA targeting the 3'-UTR of PDL1 for disrupting the 3'-UTR of PDL1 described herein is from position 990 of the PDL1 sequence set forth in SEQ ID NO: 1 to The target is position 1050, positions 17368 to 17429 of the PDL1 sequence set forth in SEQ ID NO: 28, or positions 648 to 708 of the PDL1 sequence set forth in SEQ ID NO: 2. In some embodiments, the gRNAs that target the 3'-UTR of PDL1 described herein include positions 1003 to 1022 of the PDL1 sequence set forth in SEQ ID NO: 1, or the PDL1 sequence set forth in SEQ ID NO: 28. or positions 662 to 680 of the PDL1 sequence set forth in SEQ ID NO: 2. In some embodiments, the gRNAs that target the 3'-UTR of PDL1 described herein are from positions 1021 to 1040 of the PDL1 sequence set forth in SEQ ID NO: 1, or the PDL1 sequence set forth in SEQ ID NO: 28. or the sequence between positions 679 and 698 of the PDL1 sequence set forth in SEQ ID NO: 2. In some embodiments, a gRNA targeting the 3'-UTR of PDL1 described herein comprises a sequence downstream of the 3'-UTR of PDL1 on the opposite strand (e.g., at least 5 nucleotides, at least 10 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, or more downstream ) to target. In some embodiments, the gRNAs that target the 3'-UTR of PDL1 to disrupt the 3'-UTR of PDL1 described herein are AGAGGAAGGAATGGGCCCGT (SEQ ID NO: 13), TCGGGGCTGAGCGTGACAAG (SEQ ID NO: 14) ), or a target sequence comprising the nucleotide sequence of TCTTCTTGGTATGGTCCTAA (SEQ ID NO: 15). In some embodiments, a Cas protein (e.g., Cas9), a gRNA targeting a target sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15, or one or more nucleic acids encoding a component thereof, is administered to the stem cell. delivery results in deletion of the 3'-UTR of PDL1.

[0212] In some embodiments, gRNAs for use in the gene editing systems disclosed herein further include a scaffold sequence. The scaffold sequence can include a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and / or a sequence of any tracrRNA extension sequence. A scaffold sequence may be linked to the 5' and / or 3' end of the spacer sequence. In some embodiments, the scaffold sequence is linked to the 3' end of the spacer sequence. In other embodiments, the scaffold sequence is linked to the 5' end of the spacer sequence.

[0213] In some embodiments, gRNA for use in the gene editing systems disclosed herein consists of, or consists essentially of, one of the following scaffold nucleotide sequences (e.g., up to 20 extra nucleotides at the 5' and / or 3' ends of the sequence), or consisting of: (i) acccagcctgacaccaaatttaGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 16); (ii) tactaaaaggcagcctcctagaGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 17); (iii) attggctaccttggttggatgaGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAG GCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 18); (iv) gacagctggctatccaggattcGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 19); (v) acttgcaggaggtgagggattaGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 20); (vi) attagggaatgcagactctgggGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 21); (vii) tgggtgagattagaggccactgGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACA AGGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 22); (viii) tgcttcctcccttgtctccctaGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAG GCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 23); (iv) tggcatatgagaaaagtcacagGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 24); and (x) ccctattcttttgatatactccGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 25).

[0214] In some embodiments, the gRNA for the Cas12i protein comprises a direct repeat sequence that includes a stem-loop structure proximal to the 3' end (immediately adjacent to the spacer sequence). In some embodiments, the gRNA against the Cas12i protein includes a stem loop proximal to the 3' end, and the stem is 5-8 nucleotides long. In some embodiments, the type V-I RNA guide is a direct repeat sequence that includes the sequence 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO: 26) or 5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO: 27) proximal to the 3' end. , where N refers to any nucleobase. In some embodiments, the type V-I RNA guided direct repeat comprises a sequence proximal to the 3' end, where N refers to any nucleobase.

[0215] Since the above gRNA sequences are RNA sequences, it is understood that any T (thymine) in the sequence referring to gRNA would refer to a U (or uracil) in the context of an RNA molecule. Sequences containing T (thymine) herein include both DNA and RNA molecules (where T refers to U).

[0216] Furthermore, a single gRNA molecule can be free of uracil at the 3' end of the gRNA sequence. A gRNA can include one or more uracils at the 3' end of the gRNA sequence. For example, a gRNA can contain one uracil (U) at the 3' end of the gRNA sequence. A gRNA can contain two uracils (UU) at the 3' end of the gRNA sequence. gRNA can contain three uracils (UUU) at the 3' end of the gRNA sequence. gRNA can contain four uracils (UUUU) at the 3' end of the gRNA sequence. A gRNA can contain five uracils (UUUUU) at the 3' end of the gRNA sequence. A gRNA can contain six uracils (UUUUUU) at the 3' end of the gRNA sequence. A gRNA can contain seven uracils (UUUUUUU) at the 3' end of the gRNA sequence. The gRNA can include 8 uracils (UUUUUUUU) at the 3' end of the gRNA sequence.

[0217] In some embodiments, the gene editing systems disclosed herein can include nucleic acids (eg, vectors) encoding gene editing system components or viral particles that include the same. In some embodiments, the gene editing system includes one nucleic acid capable of producing all components of the gene editing system, including a nuclease and one or more gRNAs. In other examples, the gene editing system includes two or more nucleic acids.

[0218] The nucleic acid (or at least one nucleic acid in the set of nucleic acids) may be a vector, such as a retroviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, and a viral vector, such as a herpes simplex virus (HSV) vector. good.

[0219] In some examples, a gene editing system can include one or more viral particles that carry genetic material to produce components of a gene editing system as disclosed herein. A viral particle (eg, an AAV particle) can include one or more components (or an agent for producing one or more components) of a gene editing system (eg, as described herein). Viral particles (or virions) contain a nucleic acid encoding the viral genome and a protein coat (or capsid). In some instances, the virus particle further comprises a lipid envelope surrounding the protein shell.

[0220] In some instances, the viral particle contains a nucleic acid capable of producing all components of a gene editing system, including a nuclease and one or more gRNAs. In other examples, the viral particle includes a nucleic acid capable of producing one or more components of a gene editing system. For example, a viral particle may include a nuclease and a nucleic acid capable of producing gRNA. Alternatively, the viral particle may contain a nucleic acid capable of producing one or more gRNAs. In another example, a viral particle may contain a nucleic acid capable of producing only one of the nucleases or one of the gRNAs.

[0221] Viral particles described herein may include any known in the art, including, but not limited to, retroviral particles, adenoviral particles, adeno-associated virus (AAV) particles, or herpes simplex virus (HSV) particles. virus particles. In some embodiments, the viral particles are AAV particles. In some embodiments, the AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, e.g., U.S. Patent No. 9,790,472, incorporated herein by reference in its entirety). , AAVrh74 (see, eg, US Patent Application Publication No. 2015 / 0111955, incorporated herein by reference in its entirety), or an AAV9 vector, where the number following AAV indicates the AAV serotype. Any variation of AAV vectors or their serotypes, such as self-complementary AAV (sc AAV) vectors, are encompassed by the general term AAV vectors, such as AAV1 vectors. See, for example, McCarty et al., Gene Ther. 2001;8:1248-54, Naso et al., BioDrugs 2017;31:317-334, and the references cited therein for a detailed discussion of various AAV vectors. Please refer to the following.

[0222] In some embodiments, the set of viral particles includes two or more gene editing systems. In some embodiments, each virus particle in the set of virus particles is an AAV particle. In other embodiments, the set of viral particles includes two or more types of viral particles (e.g., retroviral particles, adenoviral particles, adeno-associated virus (AAV) particles, or herpes simplex virus (HSV) particles). .

[0223] In some embodiments, the gRNA used in accordance with the present disclosure is synthetic and / or chemically modified and transferred to stem cells via methods known in the art (e.g., via transfection or lipid nanoparticles). can be delivered to. Lipid nanoparticles (LNPs) are a known means for the delivery of nucleotide and protein cargoes and can be used for the delivery of guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNP delivers a nucleic acid, a protein, or a nucleic acid along with a protein. In some embodiments, the gene editing system includes one or more ribonucleoproteins including a Cas protein (eg, a Cas9 protein or a Cas12i2 protein) and a guide RNA. In some embodiments, the ribonucleoprotein is administered to any of the cells disclosed herein (eg, any of the stem cells disclosed herein) by lipid nanoparticles. In some embodiments, the present disclosure provides a nucleic acid encoding an endonuclease (e.g., a Cas9 or Cas12i2 protein) and a nucleic acid encoding one or more gRNAs, which can be transported by the present invention by lipid nanoparticles. Any of the cells disclosed herein (eg, any of the stem cells disclosed herein) are optionally administered.

[0224] In some embodiments, the gRNA is chemically modified. gRNAs that contain one or more modified nucleosides or nucleotides are called "modified" gRNAs or "chemically modified" gRNAs and are used in place of or in addition to the canonical A, G, C, and U residues. represents the presence of one or more non-naturally occurring and / or naturally occurring components or constituents. In some embodiments, modified gRNAs are synthesized with non-canonical nucleosides or nucleotides and are referred to herein as "modified." Modified nucleosides and nucleotides may include one or more of the following: (i) modification of one or more of one or more of the unlinked phosphate oxygens and / or the linked phosphate oxygens in the phosphodiester backbone linkage; For example, substitutions (exemplary backbone modifications); (ii) modifications of ribose sugar constituents, e.g. the 2' hydroxyl on the ribose sugar, such as substitutions (exemplary sugar modifications); (iii) "dephospho" linkers. wholesale substitution of the phosphate moiety (an exemplary backbone modification); (iv) modification or substitution of naturally occurring nucleobases, including non-canonical nucleobases (an exemplary base modification); (v) ribose-phosphorus substitution or modification of the acid backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, such as removal, modification or substitution of a terminal phosphate group, or a moiety, cap or linker; conjugation (such 3' or 5' cap modifications may include sugar and / or backbone modifications); and (vii) sugar modifications or substitutions (exemplary sugar modifications).

[0225] Chemical modifications such as those described above can be combined to provide modified gRNAs that include nucleosides and nucleotides (collectively "residues") with two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase, or a modified sugar and a modified phosphodiester. In some embodiments, all bases of the gRNA are modified, eg, all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 3' end of the RNA.

[0226] In some embodiments, the gRNA includes 1, 2, 3 or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%) of the positions in the modified gRNA , at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) , a modified nucleoside or nucleotide.

[0227] Unmodified nucleic acids are susceptible to degradation by, for example, intracellular nucleases or nucleases present in serum. For example, nucleases hydrolyze phosphodiester bonds in nucleic acids. Thus, in one aspect, the gRNAs described herein can include one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a cell population both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, and is accompanied by the expression and release of cytokines, particularly the induction of interferons, and cell death.

[0228] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygens with different substituents. Additionally, modified residues, eg, those present in a modified nucleic acid, can include wholesale substitution of an unmodified phosphate moiety with a modified phosphate group, as described herein. In some embodiments, modifications of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with an asymmetric charge distribution.

[0229] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom of the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the atoms or groups of atoms mentioned above can make the phosphorus atom chiral. Stereoisomeric phosphorus atoms can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate). . Substitutions can occur at either or both of the linking oxygens.

[0230] The phosphate group can be replaced with a non-phosphorus-containing connector in certain backbone modifications. In some embodiments, charged phosphate groups may be replaced with neutral moieties. Examples of moieties that can substitute the phosphate group include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal. , formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0231] Scaffolds that mimic nucleic acids can also be constructed in which phosphate linkers and ribose sugars are replaced with nuclease-resistant nucleosides or nucleotide surrogates. Such modifications may include backbone and sugar modifications. In some embodiments, the nucleobases may be tethered by a surrogate backbone. Examples include, but are not limited to, morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0232] Modified nucleosides and modified nucleotides may contain one or more modifications to the sugar group, ie, sugar modifications. For example, the 2' hydroxyl group (OH) can be modified, eg, substituted with a number of different "oxy" or "deoxy" substituents. In some embodiments, modification of the 2' hydroxyl group can enhance the stability of the nucleic acid because the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. .

[0233] Examples of modifications of the 2' hydroxyl group include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG) ,0(CH 2 CH 20 )nCH 2 CH 20 R, where R can be, for example, H or optionally substituted alkyl, and n is an integer from 0 to 20 (for example, 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1~4, 1~8, 1~10, 1~16, 1~20, 2~4, 2~8, 2~10, 2~16, 2~20, 4~8, 4~10, 4~ 16, and 4 to 20)). In some embodiments, the 2' hydroxyl group modification can be 2'-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluorine modification that replaces the 2' hydroxyl group with fluoride. In some embodiments, the 2' hydroxyl group modification is "locked" in which the 2' hydroxyl can be linked to the 4' carbon of the same ribose sugar, e.g., by a Ci-e alkylene or Ci-e heteroalkylene bridge. Nucleic acids (LNA), where exemplary bridges include methylene, propylene, ether, or amino bridges; O-amino (where amino is, for example, N3 / 4; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino), and aminoalkoxy, 0(CH2)n-amino, (where amino is, for example, N3 / 4; alkylamino , dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification may include an "unlocked" nucleic acid (UNA) in which the ribose ring lacks a C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CH2OCH3, eg, a PEG derivative).

[0234] "Deoxy" 2' modifications include hydrogen (i.e., a deoxyribose sugar, e.g., the overhanging moiety of a partial dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (where amino is For example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-amino (where amino is e.g. cyano; mercapto; alkylthioalkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted, for example with amino as described herein. Sugar modifications can include sugar groups that include one or more carbons with a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, modified nucleic acids may include nucleotides that include, for example, arabinose as sugar. Modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified with one or more of the constituent sugar atoms. Modified nucleic acids may also include one or more sugars that are in the L form, such as L-nucleosides.

[0235] The modified nucleosides and nucleotides described herein that can be incorporated into modified nucleic acids can include modified bases, also referred to as nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or completely substituted to provide modified residues that can be incorporated into modified nucleic acids. Nucleotide nucleobases may be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. In some embodiments, nucleobases can include, for example, natural and synthetic derivatives of bases.

[0236] In embodiments employing dual guide RNAs, each of the crRNA and tracr RNA can include modifications. Such modifications may be present at one or both ends of crRNA and / or tracr RNA. In embodiments involving sgRNAs, one or more residues at one or both ends of the sgRNA may be chemically modified, and / or internal nucleosides may be modified, and / or the entire sgRNA may be chemically modified. Certain embodiments include modifications at the 5' end. Certain embodiments include 3' end modifications. In some embodiments, the modification includes a 2'-O-methyl modification.

[0237] Another chemical modification that has been shown to affect the nucleotide sugar ring is halogen substitution. For example, 2'-fluoro (2'-F) substitutions on the nucleotide sugar ring can increase oligonucleotide binding affinity and nuclease stability. Includes 2'-fluoro (2'-F) modifications. A phosphorothioate (PS) linkage or bond refers to a bond in which sulfur is substituted for one non-bridging phosphate oxygen of a phosphodiester bond, eg, in a bond between nucleotide bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides are also referred to as S-oligos.

[0238] Abasic nucleotide refers to a nucleotide lacking a nitrogenous base. A reverse base refers to one having a bond opposite to the normal 5'-3' bond (ie, either a 5'-5' bond or a 3'-3' bond).

[0239] Abasic nucleotides can be attached in reverse bonds. For example, an abasic nucleotide can be attached to a terminal 5' nucleotide via a 5' to 5' linkage, or an abasic nucleotide can be attached to a terminal 3' nucleotide via a 3' to 3' linkage. can be combined with A reverse abasic nucleotide at either the terminal 5' or 3' nucleotide may also be referred to as a reverse abasic end cap.

[0240] In some embodiments, one or more of the first 3, 4, or 5 nucleotides of the 5' end and one or more of the last 3, 4, or 5 nucleotides of the 3' end are modified. Ru. In some embodiments, the modifications include 2'-0-Me, 2'-F, reverse abasic nucleotides, PS linkages, or other modifications known in the art to increase stability and / or performance. It is a nucleotide modification.

[0241] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked with a phosphorothioate (PS) bond. In some embodiments, the first three nucleotides of the 5' end and the last three nucleotides of the 3' end comprise 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the first three nucleotides of the 5' end and the last three nucleotides of the 3' end comprise 2'-fluoro (2'-F) modified nucleotides.

[0242] In some embodiments, the methods of producing hypoimmune cells described herein include knockout of a target polynucleotide sequence or a portion thereof (e.g., 3' of an immunosuppressive factor, B2M, and / or CIITA gene). -Knockout of UTR). In some embodiments, the methods of producing hypoimmune cells described herein result in a knock-in of a target polynucleotide sequence or a portion thereof (eg, a knock-in of an immunosuppressive factor or an anti-CRISPR protein). In some embodiments, the methods can be performed in vitro, in vivo, or ex vivo for both treatment and research purposes. In some embodiments, any genetic modification described herein is a homozygous modification. In some embodiments, any genetic modification described herein is a heterozygous modification.

[0243] In some embodiments, the methods of producing hypoimmune cells described herein are performed using the CRISPR / Cas system. In some embodiments, CRISPR / Cas systems can modify target polynucleotides with high efficiency. In certain embodiments, the modification efficiency is at least about 5%. In certain embodiments, the modification efficiency is at least about 10%. In certain embodiments, the modification efficiency is about 10% to about 80%. In certain embodiments, the efficiency of modification is between about 30% and about 80%. In certain embodiments, the modification efficiency is about 50% to about 80%. In some embodiments, the modification efficiency is about 80% or greater. In some embodiments, the modification efficiency is about 85% or greater. In some embodiments, the modification efficiency is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. % or more. In some embodiments, the modification efficiency is equal to about 100%. [Example]

[0244] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. Example 1. Design and validation of PDL1 3’ UTR-targeted CRISPR / Cas9 knockout construct PDL1 has been shown to play a major role in suppressing the adaptive immune system. Normal activity of PDL1 plays a role in suppressing excessive stimulation of immune cells (CD8+ / CD4+ T cells, etc.). Therefore, PDL1 is a therapeutic target in stem cell research: overexpression of PDL1 or reduction of restricted expression of PDL1 may promote effective stem cell engraftment by inhibiting deleterious immune responses. The present disclosure describes, in part, the ability to manipulate the endogenous PDL1 3'-UTR to induce overexpression of the PDL1 gene, either constitutively or in response to specific cues (e.g., cytokines such as interferon-gamma). and how to promote it.

[0245] Manipulating the endogenous PDL1 locus provides a potential solution to several problems encountered with traditional transgene knock-in paradigms. In particular, traditional transgene knock-in approaches are susceptible to epigenetic silencing of the knock-in transgene. The present disclosure addresses this particular problem by using a representative gene editing system, namely the CRISPR / Cas9 system, to excise a large portion of the endogenous 3'UTR of the endogenous PDL1 gene. PDL1 3' UTR has a known post-transcriptional regulatory function that regulates the subsequent translation of PDL1 mRNA (for example, as described in Kataoka et al., Nature, 534, 401-418, 2016). Specific disruption of the 3'UTR of the endogenous PDL1 gene by the CRISPR / Cas9 system allows for increased expression of the PDL1 gene in cell types of interest (e.g., stem cells and stem cell derivatives).

[0246] To identify effective CRISPR / Cas9 knockout constructs to excise the 3'UTR of the endogenous PDL1 gene, human embryonic cells (hESCs) were incubated with CRISPR guide RNA and Cas9 endpoint targeting the PDL1 3' UTR or NLRC5. Nucleofection was performed with nuclease (3 μL of guide RNA (300 pmol) was mixed with 2 μL of Cas9 (40 pmol) at a ratio of 7.5:1). Cells were cultured for 2 days and then stimulated with IFNγ for 3 days. Analysis of PDL1 expression levels after IFNγ stimulation showed subtle upregulation of PDL1 gene expression. The guide RNA target sequence of PDL1 3'-UTR is shown in Table 1.

[0247] [Table 1]

[0248] Clones obtained from the previous experiment were then selected for further analysis. Twelve clones were selected from a 96-well plate and DNA was extracted (Lucigen DNA Quick Extract). The DNA of each of the 12 clones was analyzed for the presence of the 3'UTR of the endogenous PDL1 gene to determine whether the 3'UTR had been excised in any of the clones. Primers spanning the 3'UTR of the PDL1 gene were used in PCR assays using extracted DNA. Among the clones created, three clones (#12, #19, #25) were found to have a disrupted 3'UTR of the endogenous PDL1 gene.

[0249] These three clones were retested in stem cell conditions for global dysregulation of the PDL1 gene without INFγ stimulation. Surprisingly, PDL1 gene expression was upregulated in all three clones.

[0250] Example 2. Confirmation of PDL1 overexpression effect To confirm the effect of three clones containing CRISPR / Cas9 constructs in excising the 3'UTR and increasing the expression of endogenous PDL1 gene, wild-type cells, B2M / CIITA double knockout (DKO) cells, and three PDL1 3'UTR deletion clones (#12, #19, #25) were differentiated into endothelial cells and mixed with human CD8+ T cells to assay for CD69 surface expression (Figure 1A). Medium alone (negative control) did not activate CD69 surface expression, whereas medium containing CD3 / CD28 activation beads (positive control) activated CD69 surface expression. Wild-type cells appeared to activate surface expression of CD69, whereas DKO cells did not. Surprisingly, disruption of the PDL1 3'-UTR decreased T cell activation in all three clones, as measured by decreased activation of CD69 surface expression. To further confirm the cell composition used in Figure 1A, unstimulated wild-type cells, B2M / CIITA DKO cells, and three PDL1 3' UTR deletion clones were probed for HLA-I expression (Fig. 1B). Three PDL1 3' UTR deletion clones expressed HLA-I, whereas B2M / CIITA DKO cells did not.

[0251] To further confirm the PDL1 overexpression effect, purified human CD8+ T cells were isolated from B2M / CIITA DKO clones (DKO #18, DKO #46, and DKO #64) or three PDL1 3' UTR deletion clones (#12). , #19, and #25) and assessed T cell activation by assaying CD69 surface expression (Figure 2). The three PDL1 3' UTR deletion clones appeared to reduce T cell activation as effectively as the DKO clones in this experiment.

[0252] To determine whether increased PDL1 expression after 3'UTR excision requires stimulation, wild-type cells and PDL1 3'UTR deletion clones were differentiated into endothelial cells and stimulated by cytokines such as IFN-γ. PDL1 surface expression was probed (measurement of mean fluorescence intensity (MFI) by flow cytometry) in the presence and absence of stimulation (Figure 3). In the absence of stimulation, PDL1 surface expression was similar between wild-type cells and PDL1 3' UTR deletion clones. When co-cultured with CD8+ T cells, PDL1 3' UTR deletion clones showed modest upregulation of PDL1 surface expression. When exposed to INFγ stimulation, PDL1 3' UTR deletion clones showed a greater induction of PDL1 surface expression compared to wild-type cells.

[0253] To examine whether PDL1 3' UTR deletion protects cells from T cell-mediated cell death, purified human CD8+ T cells were co-incubated with wild-type cells, B2M / CIITA DKO clones, and PDL1 3' UTR deletion clones. were co-cultured (Figure 4). Wild-type cells were susceptible to T-cell-mediated cell death, and nearly all cells underwent cell death. Three of the four DKO clones were resistant to T cell-mediated cell death. Surprisingly, all PDL1 3' UTR deletion clones were resistant to T cell-mediated cell death.

Claims

1. An isolated stem cell containing a disruption in the 3'-untranslated region (3'-UTR) of an allele encoding an immunosuppressive factor.

2. The isolated stem cell of claim 1, wherein the disruption comprises a deletion, insertion, translocation, inversion, or substitution in the 3'-UTR.

3. 2. The isolated stem cell of claim 1, wherein the disruption reduces binding of the 3'-UTR to endogenous RNA-binding proteins and / or microRNAs.

4. 2. The isolated stem cell of claim 1, wherein the immunosuppressive factor is selected from the group consisting of PDL1, CD47, HLA-G, and combinations thereof.

5. The isolated stem cell of claim 1, wherein the disruption in the 3'-UTR results in increased expression of an immunosuppressive factor.

6. The isolated stem cell of claim 1 , wherein the immunosuppressive factor is PDL1.

7. The disruption reduces binding of one or more endogenous microRNAs to the PDL1 3'-UTR, and optionally, one or more of the endogenous microRNAs is selected from the group consisting of miR-34a, miR-140, miR-200a, miR-200b / c, miR-142, miR-340, miR-383, miR-424(322), miR-338-5p, miR-324-5p, miR- 152, miR-200b, miR-138-5p, miR-195, miR-16, miR-15a, miR15b, miR-193a-3p, miR-497-5p, miR-33a, miR17-5p, miR-155, and miR-513.

8. 2. The isolated stem cell of claim 1, wherein the disruption results in a deletion of 1 to 7 nucleotides in one or more of the PDL1 3'-UTR sequences set forth in any one of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 45, 48, 36, 58, 59, 61, 63, 65, 67, 69, 71, and 73.

9. 2. The isolated stem cell of claim 1, wherein the disruption results in a deletion of 1 to 24 nucleotides in one or more of the PDL1 3'-UTR sequences set forth in any one of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 44, 47, 57, 60, 62, 64, 66, 68, 70, and 72.

10. The isolated stem cell according to claim 1, wherein the immunosuppressive factor is HLA-G.

11. 11. The isolated stem cell of claim 10, wherein the disruption reduces binding of one or more endogenous microRNAs to the HLA-G 3'-UTR, and optionally, the one or more endogenous microRNAs are selected from the group consisting of miR-133A, miR-148A, miR-148B, miR-152, miR-548q, and / or miR-628-5p.

12. The isolated stem cell of claim 11, wherein the disruption results in a deletion of at least five consecutive nucleotides starting at and including position +2961 of the HLA-G 3'-UTR, and / or an insertion of at least five nucleotides at position +2961.

13. The isolated stem cell of claim 11, wherein the disruption is in the HLA-G 3'-UTR sequence set forth in SEQ ID NO:

74.

14. The isolated stem cell of claim 13, wherein the disruption results in a deletion of at least one nucleotide of the HLA-G 3'-UTR sequence set forth in SEQ ID NO:

75.

15. 14. The isolated stem cell of claim 13, wherein the disruption results in one or more mutations selected from C120G, G252C, A297G, and / or C306G in the HLA-G 3'-UTR sequence set forth in SEQ ID NO:

74.

16. 2. The isolated stem cell of claim 1, further comprising an insertion into a safe harbor locus of a sequence encoding CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, C1 inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9.

17. 2. The isolated stem cell of claim 1, which has reduced expression of MHC-I and MHC-II human leukocyte antigens (HLA) compared to wild-type stem cells of the same cell type.

18. 18. The isolated stem cell of claim 17, wherein the reduced expression of MHC-I HLA is due to a disruption in an allele encoding beta-2 microglobulin (B2M).

19. 18. The isolated stem cell of claim 17, wherein the reduced expression of MHC-II HLA is due to a disruption in an allele encoding class II major histocompatibility complex transactivator (CIITA).

20. The isolated stem cell of claim 1 , which is an embryonic stem cell.

21. The isolated stem cell of claim 1, which is a pluripotent stem cell.

22. A cell differentiated from an isolated stem cell described in any one of claims 1 to 21.

23. 23. The cell of claim 22, wherein the cell is selected from the group consisting of fibroblasts, endothelial cells, definitive endoderm cells, primitive gut cells, pancreatic progenitor cells, pancreatic endocrine cells, pancreatic islet cells, stem cell-derived beta cells, stem cell-derived alpha cells, stem cell-derived delta cells, stem cell-derived enterochromaffin (EC) cells, insulin-producing cells, insulin-positive beta-like cells, hematopoietic stem cells, hematopoietic progenitor cells, muscle cells, satellite stem cells, hepatocytes, neurons, or immune cells.

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