Non-immunogenic engineered tissue and methods of producing and using the same

Abstract

To provide a method of producing a non-immunogenic engineered tissue from pluripotent stem cells.SOLUTION: Provided is a method, in which pluripotent stem cells are deficient of endogenous MHC class I molecules presented on a cell surface of the pluripotent stem cell and comprises an immunomodulatory protein on their surface. The method comprises forming an engineered tissue in the presence of at least one cell type that is essential for a function of the engineered tissue under conditions that allow formation of the engineered tissue, and the at least one cell type has been obtained by differentiation of the pluripotent stem cells into the at least one cell type, thereby rendering the engineered tissue to be non-immunogenic to a recipient of the engineered tissue.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from European Patent Application No. 18183294.0, filed July 13, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] TECHNICAL FIELD OF THEINVENTION The present invention provides a method for producing non-immunogenic engineered tissue from pluripotent stem cells, the pluripotent stem cells lacking MHC class I molecules and containing immunomodulatory proteins on their surface, the method comprising forming the engineered tissue in the presence of at least one cell type essential for the function of the engineered tissue under conditions that allow the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue, the at least one cell type having been obtained by inducing differentiation of the pluripotent stem cells into the at least one cell type essential for the function of the engineered tissue under conditions that also allow the formation of the engineered tissue. The present invention further relates to engineered tissue, pharmaceutical compositions comprising the engineered tissue, medical treatments using the engineered tissue, and uses of the engineered tissue. [Background technology]

[0003] The shortage of suitable organs or tissues to replace dysfunctional organs or tissues remains a major problem in modern medicine, especially considering that the supply of organ donations is insufficient to meet medical need and cannot be adequately planned.

[0004] One approach to overcome this is to engineer pluripotent stem cells, particularly human pluripotent stem cells (hPSCs), into functional tissues. This field has seen a variety of very promising results recently. For example, International Patent Application WO 2015 / 025030 describes a method for producing engineered cardiac muscle, while International Patent Application WO 2015 / 040142 describes an improved differentiation protocol into cardiac tissue. Rao et al, “Engineering human pluripotent stem cells into functional skeletal muscle tissue” Nature Communications, (2018) 9:126 derives induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. Rao et al. report that in 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells that endogenously express Pax7. The review by Lancaster and Knoblich, "Organogenesis in a dish: Modeling development and disease using organoid technologies" Science 345, 1247125 (2014) describes organoids (stem cell-derived three-dimensional cultures) derived from human PSCs, particularly of intestinal, renal, brain, and retinal tissues. Similarly, the review by Llonch et al., "Organoid technology for retinal repair" Developmental Biology 433 (2018) 132-143, discusses PSC-derived retinal organoids as an important tool for generating retinal tissue in vitro, widely used to generate large numbers of photoreceptors, which can be further developed toward potential cell-based therapies.Finally, Pagliuca et al, "Generation of Functional Human Pancreatic b Cells In Vitro" Cell 159, 428-439 (2014) report a scalable differentiation protocol capable of generating hundreds of millions of glucose-responsive b cells from hPSCs in vitro.

[0005] However, the methods described above, while yielding, for example, functional cardiac or skeletal tissue, still face the challenge of graft rejection if the pluripotent stem cells utilized are derived from an allogeneic donor and / or are not histologically compatible.

[0006] Therefore, there is a need not only for functional tissue or organoids for therapeutic applications in general, such as functional cardiac tissue, but also for improved functional tissue or organoids, such as improved cardiac tissue that originates from an allogeneic donor but is not rejected in the recipient.The present invention aims to solve this problem. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Application WO 2015 / 025030 [Patent Document 2] International Patent Application WO 2015 / 040142 [Non-patent literature]

[0008] [Non-Patent Document 1] Rao et al, “Engineering human pluripotent stem cells into functional skeletal muscle tissue” Nature Communications, 9:126 (2018) [Non-Patent Document 2] Lancaster and Knoblich, “Organogenesis in a dish: Modeling development and disease using organoid technologies” Science 345, 1247125 (2014) [Non-Patent Document 3] Llonch et al “Organoid technology for retinal repair” Developmental Biology 433, 132-143 (2018) [Non-Patent Document 4] Pagliuca et al, “Generation of Functional Human Pancreatic cells In Vitro” Cell 159, 428-439 (2014) Summary of the Invention

[0009] The problem is solved by the subject matter as defined in the claims. The invention provides a method for producing non-immunogenic engineered tissue from pluripotent cells, the engineered tissue, a pharmaceutical composition comprising the engineered tissue, a medical treatment using the engineered tissue, and uses of the engineered tissue.

[0010] Accordingly, the present invention relates to a method of producing a non-immunogenic engineered tissue from pluripotent stem cells, wherein the pluripotent stem cells lack endogenous MHC class I molecules displayed on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on their surface, the method comprising forming the engineered tissue in the presence of at least one cell type that is essential for the function of the engineered tissue, under conditions that allow the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue, wherein the at least one cell type has been obtained by differentiation of the pluripotent stem cells into the at least one cell type.

[0011] The invention also relates to a method of producing a non-immunogenic engineered tissue from pluripotent stem cells, wherein the pluripotent stem cells lack endogenous MHC class I molecules displayed on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on their surface, wherein the method comprises inducing differentiation of the pluripotent stem cells into at least one cell type that is essential for the function of the engineered tissue, under conditions that also allow for the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue.

[0012] In embodiments of the invention, the engineered tissue (a) is not recognized as allogeneic by the recipient's effector T cells, and / or (b) is resistant to NK-mediated lysis. Preferably, the engineered tissue does not bind anti-HLA antibodies, and preferably, the tissue does not bind anti-HLA-A or anti-HLA-B antibodies.

[0013] The immunomodulatory protein may be a single-chain fusion HLA class I protein, where more preferably the single-chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. Most preferably, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C, or the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F.

[0014] In an embodiment of the invention, the pluripotent stem cells further express a target peptide antigen presented by a single chain fusion HLA class I protein on the pluripotent cell surface, and more preferably, the target peptide antigen is covalently linked to the single chain fusion HLA class I protein, and wherein the target peptide antigen may comprise the sequence VMAPRTLFL (SEQ ID NO: 1).

[0015] In an embodiment of the invention, essentially all copies of the β-microglobulin 2 gene are disrupted in the pluripotent stem cells.

[0016] In an embodiment of the invention, the method includes forming an engineered tissue in the presence of at least one second cell type that forms part of the issue. The second cell type that forms part of the engineered tissue depends on the type of tissue being engineered (e.g., when engineered myocardial or hepatic tissue is being formed) and can be, for example, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, adipocytes, reticular cells, or mesenchymal stem cells.

[0017] The engineered tissue may be selected from the group consisting of cardiac tissue, liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, skeletal muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue, retinal tissue and vascular tissue.

[0018] Preferably, in one embodiment of the method of the present invention, the engineered tissue is cardiac tissue, wherein the method further comprises the steps of: (i) injecting pluripotent stem cells into a culture medium containing an effective amount of (a) BMP4, activin A, FGF2, a GSK3 inhibitor, and (b) 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement as described in (i), thereby inducing cardiac differentiation of the cells; and (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of a serum-free supplement as described in (i), under mechanical stimulation, thereby promoting cardiac maturation.

[0019] In one embodiment of the method of the present invention, the formation of the tissue is carried out in the presence of a hydrogel, preferably an extracellular matrix protein, such as a fibrin hydrogel or a collagen hydrogel, most preferably a collagen hydrogel.

[0020] In one embodiment of the method of the invention, the method further comprises the step of: (iv) providing a serum-free reconstitution mixture in one or more forms, the reconstitution mixture comprising: (a) serum-free minimal essential medium; (b) 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml L-carnitine HCl, 0.1-10 μg / ml hydrocortisone, 0.05-5 μl / ml fatty acid supplement, 0.0001-0.1 μg / ml triiodo-L-thyronine (T3), and 0.2-2 and (c) the cells obtained in step (iii) and a cell type forming part of an engineered tissue, preferably human non-muscle cells, optionally wherein the cells forming part of the engineered tissue are derived from pluripotent stem cells, and 20-80% of the total cell mixture are the cells obtained in step (iii); the reconstituted mixture has a pH of 7.2-7.6; (v) culturing the serum-free reconstituted mixture in said one or more molds, thereby causing the serum-free reconstituted mixture to be compacted for at least 15 minutes; (vi) culturing the mixture obtained in step (v) in said one or more molds in serum-free EHM culture medium until the mixture in said one or more molds is compacted to at least 50% of its original thickness, wherein the EHM culture medium comprises: (a) 0.5-3 mmol / L Ca 2+ (b) (i) a basal medium comprising a serum-free supplement as defined in (b); (c) 0.5-10 mmol / L L-glutamine; (d) 0.01-1.0 mmol / L ascorbic acid; (e) 1-100 ng / ml IGF-1; and (f) 1-10 ng / ml TGFβ1; (vii) culturing the mixture obtained in step (iii) in serum-free EHM culture medium as defined in steps (iii) (a)-(f) under mechanical stretch, thereby forming a force-generating engineered cardiac tissue.

[0021] Preferably, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells.

[0022] Preferably, the pluripotent stem cells are pluripotent stem cells of primate origin, preferably human pluripotent stem cells.Preferably, the pluripotent stem cells.Preferably, the pluripotent stem cells are ND50039 in the NINDS Human Cell and Data Repository.

[0023] Preferably, the method of the invention further comprises the step of inducing differentiation of pluripotent stem cells into at least one cell type that forms part of the engineered tissue, wherein the cells essential for the function of the engineered tissue are contacted after differentiation to form the engineered tissue. The (second) cell type that may form part of the engineered tissue may be, but is not limited to, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, adipocytes, reticular cells or mesenchymal stem cells, to name just a few illustrative examples.

[0024] Preferably, the disruption of B2M and / or the insertion of the immunomodulatory protein is mediated by an engineered nuclease. More preferably, the engineered nuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9). Most preferably, the engineered nuclease is CRISPR / Cas9 and the crRNA is TIFF2025038086000001.tif26167.

[0025] In one embodiment, pluripotent stem cells are differentiated into said at least one cell type during the formation of the engineered tissue.

[0026] The present invention also relates to engineered tissues comprising pluripotent stem cells that lack MHC class I molecules and contain immunomodulatory proteins on their surface, where the pluripotent stem cells have been differentiated into cell types that are essential for the function of the engineered tissue under conditions that also allow for the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue.

[0027] The present invention also relates to engineered tissue obtainable by the method of the present invention.The present invention further relates to engineered tissue obtainable by the method of the present invention.

[0028] Preferably, the engineered tissue further comprises an extracellular matrix biomaterial. More preferably, the extracellular matrix biomaterial is an alginate, a hydrogel, a collagen hydrogel, a fibrin hydrogel, or a synthetic matrix such as polylactic acid, polyglycolic acid, and polyglycerol sebacate (bio-rubber), and poly(octamethylene maleate (anhydride) citrate, and most preferably, the extracellular matrix biomaterial is type I collagen.

[0029] Preferably, the engineered tissue (a) is not recognized as allogeneic by the recipient's effector T cells, (b) does not bind anti-HLA antibodies, and / or (c) is resistant to NK-mediated lysis.

[0030] The present invention further relates to pharmaceutical compositions comprising the engineered tissues of the present invention.

[0031] The present invention further relates to an engineered tissue of the present invention or a pharmaceutical composition of the present invention for use in a method of treating a disease condition.

[0032] The present invention further relates to a method of treating a disease condition comprising administering to a subject in need thereof an effective amount of an engineered tissue of the present invention or a pharmaceutical composition of the present invention.

[0033] The engineered tissue or pharmaceutical composition for use according to the invention or the method of treatment according to the invention, wherein the disease state is selected from the group consisting of diabetes, an autoimmune disease, cancer, an infectious disease, myocardial infarction, heart failure, a skeletal or joint condition, osteogenesis imperfecta, burns, liver failure, kidney failure, brain injury or soft tissue injury.

[0034] The present invention further relates to the use of any engineered tissue of the present invention: (a) in an in vitro model for drug toxicity screening; and / or (b) as a research tool.

[0035] The present invention further relates to a nucleic acid comprising at least one of SEQ ID NOs: 2-7.

[0036] The present invention also relates to the use of nucleic acids to disrupt the (expression) of the B2M gene.

[0037] The present invention will be better understood with reference to the detailed description, when considered in conjunction with the drawings, the non-limiting examples and the appended claims. [The present invention 1001] 1. A method of producing a non-immunogenic engineered tissue from pluripotent stem cells, the pluripotent stem cells lacking endogenous MHC class I molecules displayed on the cell surface of the pluripotent stem cells and comprising immunomodulatory proteins on their surface; The method comprises: forming an engineered tissue in the presence of at least one cell type essential for the function of the engineered tissue under conditions that allow for the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue, wherein the at least one cell type was obtained by differentiation of pluripotent stem cells into the at least one cell type. The method comprising: [The present invention 1002] The engineered tissue comprises: (a) is not recognized as allogeneic by effector T cells; and / or (b) is resistant to NK-mediated lysis; The method of the present invention 1001. [The present invention 1003] 13. The method of claim 1001 or 1002, wherein said engineered tissue does not bind to anti-HLA antibodies. [The present invention 1004] 13. The method of claim 1001 or 1002, wherein said immunomodulating protein is a single chain fusion HLA class I protein. [The present invention 1005] The method of claim 1004, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of an HLA class I alpha chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. [The present invention 1006] 1004. The method of claim 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A. [The present invention 1007] The method according to any of claims 1004 to 1006, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201. [The present invention 1008] 1004. The method of claim 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E. [The present invention 1009] The method of any one of claims 1004 to 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G. [The present invention 1010] 1004. The method of claim 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B. [The present invention 1011] 1004. The method of claim 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C. [The present invention 1012] The method of any one of claims 1004 to 1005, wherein said single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F. [The present invention 1013] The method according to any of claims 1004 to 1012, wherein said pluripotent stem cells further express a target peptide antigen presented by a single-chain fusion HLA class I protein on the surface of said pluripotent cells. [The present invention 1014] The method of claim 1013, wherein said target peptide antigen is covalently linked to a single chain fusion HLA class I protein. [The present invention 1015] The method of any one of claims 1013 to 1014, wherein said target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO: 1). [The present invention 1016] The method of any of claims 1001 to 1015, wherein essentially all copies of the β-microglobulin 2 gene are disrupted in the pluripotent stem cells. [The present invention 1017] The method of any of claims 1001 to 1016, comprising forming the engineered tissue in the presence of at least one second cell type that forms part of the tissue. [The present invention 1018] The method of claim 1017, wherein the second cell type forming part of the engineered tissue is selected from the group consisting of fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, adipocytes, reticular cells and mesenchymal stem cells. [The present invention 1019] 9. The method of any of claims 1001 to 1018, wherein said engineered tissue is selected from the group consisting of cardiac tissue, liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, skeletal muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue, retinal tissue and vascular tissue. [The present invention 1020] wherein the engineered tissue is cardiac tissue, and the method comprises: (i) administering pluripotent stem cells to an effective amount of (a) BMP4, activin A, FGF2, GSK3 inhibitor, and (b) a serum-free supplement resulting in a final concentration of 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml L-carnitine HCl, 0.1-10 μg / ml hydrocortisone, 0.05-5 μl / ml fatty acid supplement, and 0.0001-0.1 μg / ml triiodo-L-thyronine (T3) thereby inducing mesodermal differentiation of the pluripotent stem cells; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement as described in (i), thereby inducing cardiac differentiation of the cells; and (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of a serum-free supplement as described in (i) under mechanical stimulation, thereby promoting cardiac maturation. Any of the methods of the present invention 1001 to 1019, comprising: [The present invention 1021] The method according to any of claims 1001 to 1020, wherein said formation of tissue is carried out in the presence of a hydrogel, preferably an extracellular matrix protein-containing hydrogel, such as a fibrin hydrogel or a collagen hydrogel, most preferably a collagen hydrogel. [The present invention 1022] (iv) providing a serum-free reconstitution mixture in one or more molds, the reconstitution mixture comprising: (a) serum-free minimal essential medium; (b) a serum-free supplement resulting in a final concentration of 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml L-carnitine HCl, 0.1-10 μg / ml hydrocortisone, 0.05-5 μl / ml fatty acid supplement, 0.0001-0.1 μg / ml triiodo-L-thyronine (T3), and 0.2-2 mg / ml collagen; and (c) the cells obtained in step (iii) and a cell type that forms part of the engineered tissue, preferably a human non-muscle cell optionally wherein the cells forming part of the engineered tissue are derived from pluripotent stem cells, and 20-80% of the total cell mixture are cells obtained in step (iii); the reconstituted mixture has a pH of 7.2 to 7.6; (v) incubating the serum-free reconstituted mixture in said mold or molds, thereby compressing the serum-free reconstituted mixture for at least 15 minutes; (vi) culturing the mixture obtained in step (v) in said one or more molds in a serum-free EHM culture medium until the mixture is compressed to at least 50% of its original thickness, wherein said EHM culture medium contains (a) 0.5 to 3 mmol / L Ca 2+ (b) a serum-free supplement as defined in (i)(b); (c) 0.5-10 mmol / L L-glutamine; (d) 0.01-1.0 mmol / L ascorbic acid; (e) 1-100 ng / ml IGF-1; and (f) 1-10 ng / ml TGFβ1; (vii) culturing the mixture obtained in step (iii) under mechanical stretch in serum-free EHM culture medium as defined in steps (iii) (a) to (f), thereby forming a force-generating engineered cardiac tissue. The method of any one of claims 1020 to 1021, further comprising: [The present invention 1023] The method according to any one of claims 1001 to 1022, wherein said pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells. [The present invention 1024] The method according to any of claims 1001 to 1023, wherein said pluripotent stem cells are pluripotent stem cells of primate origin, preferably human pluripotent stem cells. [The present invention 1025] The method according to any one of claims 1001 to 1024, wherein said pluripotent stem cells are prepared from CD34 positive cells isolated from umbilical cord blood. [The present invention 1026] The method according to any one of claims 1001 to 1025, wherein said pluripotent stem cells are ND-50039 of the NINDS Human Cell and Data Repository. [The present invention 1027] Further inducing differentiation of the pluripotent stem cells into at least one second cell type that forms part of the engineered tissue, wherein after differentiation, cells of the cell type essential for the function of the engineered tissue are contacted with cells of the second cell type that forms part of the engineered tissue to form the engineered tissue. Any of the methods of 1001 to 1026 of the present invention, further comprising: [The present invention 1028] The method of claim 1027, wherein the second cell type forming part of the engineered tissue is a fibroblast, an endothelial cell, a smooth muscle cell, a chondrocyte, an adipocyte, a reticular cell or a mesenchymal stem cell. [The present invention 1029] The method of any of claims 1001 to 1028, wherein the disruption of B2M and / or the insertion of the immunomodulatory protein is mediated by an engineered nuclease. [The present invention 1030] The method of the present invention, wherein the engineered nuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9). [The present invention 1031] The engineered nuclease is CRISPR / Cas9 and the crRNA is The method of the present invention 1029 or 1030, selected from the group consisting of TIFF2025038086000002.tif42146. [The present invention 1032] The method according to any of claims 1001 to 1031, wherein said pluripotent stem cells are differentiated into said at least one cell type during the formation of an engineered tissue. [The present invention 1033] An engineered tissue comprising a cell type that is essential for the function of the engineered tissue, the cell type has been obtained by differentiating a pluripotent stem cell into said cell type under conditions suitable for differentiation of the pluripotent stem cell into said cell type, the pluripotent stem cells lacking MHC class I molecules and comprising immunomodulatory proteins on their surface; thereby rendering the engineered tissue non-immunogenic to the recipient of the engineered tissue; The engineered tissue. [The present invention 1034] The engineered tissue of the present invention 1033 further comprising at least a second cell type forming part of the tissue. [The present invention 1035] An engineered tissue obtainable by any of the methods of the present inventions 1001 to 1032. [The present invention 1036] An engineered tissue obtained by any of the methods of the present inventions 1001 to 1032. [The present invention 1037] The engineered tissue of any of claims 1033 to 1036, further comprising an extracellular matrix biomaterial. [The present invention 1038] 1037. The engineered tissue of claim 1037, wherein said extracellular matrix biomaterial is an alginate, a hydrogel, a collagen hydrogel, a fibrin hydrogel, or a synthetic matrix such as polylactic acid, polyglycolic acid, and polyglycerol sebacate (bio-rubber), and poly(octamethylene maleate (anhydride) citrate, preferably said extracellular matrix biomaterial is type I collagen. [The present invention 1039] The engineered tissue of claim 1038, wherein said synthetic matrix material is polylactic acid or polyglycolic acid. [The present invention 1040] The engineered tissue comprises: (a) are not recognized as allogeneic by effector T cells; (b) does not bind to anti-HLA antibodies; and / or (c) is resistant to NK-mediated lysis; The engineered tissue of any one of claims 1033 to 1039. [The present invention 1041] A pharmaceutical composition comprising any one of the engineered tissues of the present inventions 1033 to 1040. [The present invention 1042] The engineered tissue of any of claims 1033 to 1040 or the pharmaceutical composition of claim 1041 for use in a method of treating a disease condition. [The present invention 1043] A method of treating a disease condition comprising administering an effective amount of the engineered tissue of any of claims 1033-1040 or the pharmaceutical composition of claim 1041 to a subject in need thereof. [The present invention 1044] The engineered tissue or pharmaceutical composition for use in accordance with the present invention 1042 or the method of treatment of the present invention 1043, wherein said disease state is selected from the group consisting of diabetes, an autoimmune disease, cancer, an infectious disease, myocardial infarction, heart failure, a skeletal or joint condition, osteogenesis imperfecta, burns, liver failure, kidney failure, brain injury, or soft tissue injury. [The present invention 1045] (a) in in vitro models for drug toxicity screening, and / or (b) As a research tool Use of an engineered tissue according to any one of claims 1033 to 1040. [The present invention 1046] A nucleic acid comprising at least one sequence selected from the group consisting of SEQ ID NO: 2-7. [The present invention 1047] Use of a nucleic acid of the invention 1046 to disrupt expression of the B2M gene. [Brief description of the drawings]

[0038] [Figure 1] The section of the sequence of the B2M gene surrounding exon 1 (gray, positions 201-327) is shown. The 550 base pairs shown in FIG. 1 correspond, for example, to sequence positions 4811-5360 of NCBI GenBank entry NG_012920, version NG_012920.2 dated June 3, 2018, which shows the complete gene. Underlined are the binding sites of the three crRNAs shown below the gene sequence. SEQ ID NO: 6 (B2M_CR1) binds to positions 287-299 of the complementary strand, SEQ ID NO: 5 (B2M_CR2) binds to positions 311-333 of the complementary strand, and SEQ ID NO: 7 (B2M_CR3) binds to positions 254-276. The relevant PAMs are shown in bold and italics. Also highlighted is the translation start signal ATG (double underlined). [Diagram 2]Figure 1 represents the resulting sequences of four different PSC clones after gene editing with CRISPR / Cas9 nuclease at sequence positions 201-350 of the B2M gene section shown in Figure 1. For each clone, the resulting sequences of both alleles are shown. Underlined are the binding sites of the three crRNAs: SEQ ID NO: 6 (B2M_CR1) binds to positions 287-299 of the complementary strand, SEQ ID NO: 5 (B2M_CR2) binds to positions 311-333 of the complementary strand, and SEQ ID NO: 7 (B2M_CR3) binds to positions 254-276. Also highlighted are the start codon (double underlined), the PAM (bold and italicized) and the mutations (wavy line). Deletions are marked by "-". [Figure 3-1] Flow cytometry analysis of four different PSC clones after gene editing with CRISPR / Cas9 nuclease compared to wild-type PSCs and unstained controls. Cell surface expression of B2M (APC, x-axis) and HLA (PE, y-axis) was analyzed in unstimulated PSCs (left) and PSCs stimulated with interferon-γ for 24 h (right). [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Figure 4-1]4 represents exemplary DNA sequences that allow overexpression of immunomodulatory proteins on the surface of pluripotent stem cells and can be incorporated into a suitable vector. In general, the immunomodulatory protein can comprise a functional B2M fused to an HLA gene. In an exemplary embodiment shown in FIG. 4, human B2M is fused to an HLA-E gene. The open reading frame of the "dimer" marked in grey is translated into a protein shown in SEQ ID NO: 18. This dimer comprises B2M and HLA-E fused by (G4S)4. The open reading frame of the "trimer" marked in grey is translated into a protein shown in SEQ ID NO: 20. In addition to B2M and HLA-E, this protein further comprises a target peptide antigen having the sequence MAPRTLFLGGGGSGGGGSGGGGSIQRTPK (SEQ ID NO: 21). Using such an exemplary target peptide antigen fused to a single chain B2M-HLA-E dimer can increase the stability of the complex. Thus, SEQ ID NOs: 18 and 20 are exemplary embodiments of immunomodulatory proteins. The sequences not marked in grey before and after the open reading frame are homology arms that mediate integration into the B2M gene. [Figure 4-2] This is a continuation of Figure 4-1. [Diagram 5] This figure shows the generation of HLA-E KI TC1133 hIPSC line. Figure 5(A) shows a schematic diagram of Crispr / Cas9 targeting-induced HDR-mediated HLA-E dimer and trimer knock-in in B2M KO hIPSC. Figure 5(B) shows the bright field images of untransfected hIPSC (control) and hIPSC transfected with plasmid containing HLA-E dimer and trimer donor sequence 48 hours after transfection. Figure 5(C) shows GFP-expressing hIPSC (left) and flow cytometry analysis of GFP+ cells (right) after transfection with pmaxGFP plasmid. [Figure 6A]This represents the knock-in of HLA-E upstream in the B2M locus. Figure 6 (A) shows a schematic diagram of the B2M locus and the primers encompassing the 5'-homology arm and donor sequence for PCR amplification. Agarose gel electrophoresis shows Crispr / Cas9-induced HDR-mediated gene integration in hIPSCs transfected with HLA-E dimer and trimer donor plasmids. [Figure 6B] This represents a knock-in of HLA-E upstream in the B2M locus. Figure 6(B) shows genotyping of clones for HLA-E dimers. WT: wild type, PD: plasmid DNA, HLA-E: hIPSC pool transfected with HLA-E donor plasmid. [Figure 6C] This represents a knock-in of HLA-E upstream in the B2M locus. Figure 6(C) shows genotyping for HLA-E trimer insertion. WT: wild type, PD: plasmid DNA, HLA-E: hIPSC pool transfected with HLA-E donor plasmid. [Figure 7A] 7A depicts knock-in of the entire HLA-E sequence in the B2M locus. Figure 7(A) is a schematic representation of the B2M locus, the 5' and 3'-homology arms for PCR amplification and primers encompassing the donor sequence. [Figure 7B] This represents the knock-in of the entire HLA-E sequence in the B2M locus. Figure 7(B) shows the genotyping of the clones for HLA-E dimers. [Figure 7C] This represents the knock-in of the entire HLA-E sequence in the B2M locus. Figure 7(C) shows genotyping for the HLA-E trimer insertion. [Figure 8] Figure 2. Pluripotency of HLA-E KI hIPSCs. Flow cytometry analysis of WT, B2M KO, HLA-E dimer clones #5 and 78, HLA-E trimer clones #66 and 100 for expression of pluripotency markers; OCT4A and Nanog detected in APC-A and PE-A channels, respectively. [Figure 9-1]HLA expression in HLA-E KI hIPSCs. Flow cytometry analysis of WT, B2M KO, HLA-E dimer clones #5 and 78, HLA-E trimer clones #66 and 100 for expression of B2M and HLA-E detected in APC-A and Pacific Blue-A channels, respectively. Unstained traces are shown in black. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10A] HLA expression in HLA-E KI CM. Figure 10(A) shows bright field images of hIPSCs and their subsequent differentiation into CMs expressing α-actinin, cTnT and Nuclei. Highly pure CM (right) with >90% α-actinin+ as measured by flow cytometry. Isotype control tracings are shown as empty areas. Scale bar: 50 μm. [Figure 10B-1] Figure 10(B) shows flow cytometry analysis of CM from WT, B2M KO, HLA-E dimer clones #5 and 78, HLA-E trimer clone #66 and 100 hIPSC lines for expression of B2M, HLA-B, C and HLA-E detected in APC-A, PE-A and Pacific Blue-A channels, respectively. [Figure 10B-2] This is a continuation of Figure 10B-1. [Figure 11A] 11A shows a schematic diagram of the EHM manufacturing process; hiPSC-derived cardiomyocytes (CMs) from naive GMP hiPSC line ND50039 (also known as TC1133 and available from Lonza) and from hiPSC line TC1133 genetically modified as described herein were mixed with human dermal fibroblasts in type I collagen according to Tiburcy et al. 2017 to form ring-shaped EHMs. [Figure 11B]11B depicts the hypoimmunogenic engineered human myocardium (EHM) of the present invention. Figure 11B shows the consolidation stage of the EHM in a mold for 3 days, its functional maturation under mechanical loading on a flexible stretcher for up to 4 weeks, and measurements of the force of contraction (FOC) under isometric conditions in a thermostated organ bath. [Figure 11C] Figure 11C shows the force of contraction (FOC) recorded under electrical stimulation in genetically naive (wild type) EHM and EHM containing B2M KO, HLAE dimer and trimer KI iPSC-derived cardiomyocytes. FOC responses were recorded under increasing calcium concentrations (n=4 / group). [Figure 12] 1 shows an exemplary engineered tissue that can be produced by the methods of the invention, in which cardiomyocytes and fibroblasts have been formed into cardiac tissue, also known as an "EHM patch." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention The present invention is based on the surprising finding that pluripotent stem cells lacking the expression of functional β2-microglobulin (B2M), and thus lacking MHC class I molecules, but expressing HLA-E, an immunomodulatory protein, can be used for the manufacture of engineered tissues that are non-immunogenic to the recipient of the engineered tissue. The use of pluripotent stem cells lacking MHC class I molecules and containing immunomodulatory proteins on their surface has the advantage that it provides an allogeneic engineered tissue that is, however, no longer recognized as allogeneic by the recipient's immune system. Importantly, therefore, the so obtained allogeneic engineered tissue (also referred to herein as "robotic tissue" or "stealth tissue" with respect to the recipient's immune system), obtained by stem cells being differentiated into cells essential for the function of the tissue, does not require the recipient of the tissue (patient) to be subjected to immunosuppression in order to completely avoid or at least reduce the risk of complications such as tissue rejection or graft-versus-host disease. This makes the robotic tissue of the present invention an ideal candidate for therapeutic applications of functional tissues such as engineered cardiac, hepatic, retinal or renal tissues, for example in organ or tissue replacement transplantation. In this context, the disruption of the β2-microglobulin (B2M) gene to eliminate the surface expression of MHC class I molecules to provide cells lacking MHC class I (HLA class I) was already described in 1992 by International Patent Application WO 92 / 09688 (see also International Patent Application WO 2012 / 145384), however, it is noted that this approach leaves the cells vulnerable to lysis by natural killer (NK) cells, as reported by Gornalusse et al. "HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells" (2017), Nature Biotechnology, 35(8):765-772. To address this "loss of self" response, Gornalusse et al. use forced expression of minimally polymorphic HLA-E molecules.With this approach, Gornalusse et al. generate pluripotent stem cells that can be differentiated into CD45+ hematopoietic cells that were able to avoid alloreactions and lysis by NK cells.

[0040] In the method of the present invention, pluripotent stem cells are differentiated into at least one cell type that is essential for the function of the desired engineered tissue. This differentiation can occur before the engineered tissue is formed (see, for example, Example 3 and Figure 10A in this context), or the differentiation of pluripotent stem cells can be carried out during the formation of the engineered tissue. In other words, pluripotent stem cells can be differentiated before the actual production of the engineered tissue begins, or the differentiation of pluripotent stem cells can be carried out simultaneously (contemporaneously) with the production of the engineered tissue. Such engineered tissues may contain only one cell type, e.g., insulin-producing pancreatic β-cells as described, e.g., in Rao et al, Nature Communications, (2018), supra, or induced myogenic progenitor cells (iMPCs) differentiated from hPSCs, where the iMPCs can be readily differentiated into spontaneously contracting multinucleated myotubes, which under 3D culture conditions can reproducibly form functional skeletal muscle tissue (iSKM bundles) containing aligned multinucleated myotubes that exhibit a positive force-contraction frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. In accordance with the above, it is also possible for engineered tissues to contain two or more cell types that are essential for the function of the desired engineered tissue. An illustrative example of such a tissue is pancreatic tissue, more specifically pancreatic islets, which contain alpha cells (20% of all islet cells) that produce glucagon, beta cells (about 70%) that produce insulin and amylin, delta cells (<10%) that produce somatostatin, PP cells (gamma cells or F cells) (<5%) that produce pancreatic polypeptide, and epsilon cells (<1%) that produce ghrelin. Alternatively, engineered tissues may contain two or more cell types, for example, one cell type that is essential for the function of the engineered tissue, and a supporting (second) cell type that also forms part of the (engineered) tissue. An illustrative example of such engineered tissues that contain at least two different cell types is cardiac tissue, which contains cardiomyocytes (as the first or at least one cell type) that perform the function of the tissue being primarily muscle, and fibroblasts (as the second cell type) that provide connective tissue. (See, for example, WO 2015 / 025030).In an illustrative example of such cardiac tissue formed in accordance with WO 2015 / 025030, the engineered cardiac tissue of the invention may be formed from a mixture of mixture of human cardiomyocytes and human non-myocytes, where 20-80% of the total cell mixture are cardiomyocytes (differentiated from pluripotent stem cells of the invention) and the remaining cells are non-cardiomyocytes, e.g., fibroblasts (i.e., cells of a second cell type that form part of the tissue of the invention).

[0041] Accordingly, the present invention relates to a method of producing a non-immunogenic engineered tissue from pluripotent stem cells, wherein the pluripotent stem cells lack endogenous MHC class I molecules displayed on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on their surface, the method comprising forming the engineered tissue in the presence of at least one cell type that is essential for the function of the engineered tissue, under conditions that allow the formation of the engineered tissue, rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue, wherein the at least one cell type has been obtained by differentiation of the pluripotent stem cells into the at least one cell type.

[0042] The invention further relates to a method of producing non-immunogenic engineered tissue from pluripotent stem cells, which pluripotent stem cells lack MHC class I molecules and contain immunomodulatory proteins on their surface, wherein the method comprises inducing differentiation of the pluripotent stem cells into at least one, or for example 2, 3, 4, or 5, cell types that are essential for the function of the engineered tissue, under conditions that also allow for the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue.

[0043] The term "pluripotent stem cells" (PSCs), as used herein, refers to cells that can differentiate into any cell type of the body. Thus, pluripotent stem cells offer a unique opportunity to be differentiated into essentially any tissue or organ. Currently, the most commonly utilized pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESC lines were first established by Thomson and coworkers (Thomson et al. (1998), Science 282:1145-1147). Human ESC research has recently allowed the development of a new technology to reprogram somatic cells into ES-like cells. This technology was developed by Yamanaka and coworkers in 2006 (Takahashi & Yamanaka (2006), Cell, 126:663-676). The resulting induced pluripotent cells (iPSCs) behave very similarly to ESCs and, importantly, can also differentiate into any somatic cell. Moreover, it has also been reported that parthenogenetic stem cells are suitable for EHM production in mouse models (Didie et al. (2013), J Clin Invest., 123:1285-1298); the use of human parthenogenetic stem cells according to WO 2015 / 025030 will likely give rise to human EHM. Thus, the pluripotent stem cells used herein may be selected, for example, from embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells. In the context of the present invention, these pluripotent stem cells are, however, preferably not produced using a process that involves modifying the genetic identity of the human germline or that involves the use of human embryos for industrial or commercial purposes. Preferably, the pluripotent stem cells are of primate origin, more preferably human.Suitable PSCs, including induced PSCs, can be obtained, for example, from the NIH human embryonic stem cell registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), the Stem Cell Repository of the German Center for Cardiovascular Research (DZHK), or the ATCC, to name just a few sources. Pluripotent stem cells are also available for commercial use, for example, from the NINDS Human Cell and Data Repository (https: / / stemcells.nindsgenetics.org), which is run by the US National Institute of Neurological Disorders and Stroke (NINDS) and distributes human cell resources widely to academic and industrial researchers. One illustrative example of a suitable cell line that can be used in the present invention is cell line ND50039, which is an artificial (unedited) pluripotent stem cell derived from umbilical cord blood stem cells. Further exemplary iPSC cell lines that can be used in the present invention include, but are not limited to, Gibco™'s Human Episomal iPSC Line (Order No. A18945, Thermo Fisher Scientific), or iPSC cell lines ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027 or ATCC ACS-1030 available from ATCC.Alternatively, one skilled in the art of reprogramming can easily generate suitable iPSC lines by known protocols such as those described by Okita et al, "A more efficient method to generate integration-free human iPS cells" Nature Methods, Vol.8 No.5, May 2011, pages 409-411 or by Lu et al "A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells", Biomaterials 35 (2014) 2816e2826.

[0044] As explained above, the (induced) pluripotent stem cells used in the present invention can be derived from any suitable cell type (e.g., from stem cells, e.g., mesenchymal stem cells, or epithelial stem cells, or from differentiated cells, e.g., fibroblasts) and from any suitable source (body fluid or tissue). Examples of such sources (body fluid or tissue) include umbilical cord blood, skin, gums, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., the amniotic membrane or Wharton's gel of the umbilical cord), the umbilical cord-placental junction, placenta or adipose tissue, to name just a few. In one illustrative example, the isolation of CD34-positive cells from umbilical cord blood, e.g., by magnetic cell sorting using an antibody specifically directed against CD34, followed by reprogramming, as described in Chou et al. (2011), Cell Research, 21:518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659, show that the iPSC production process can comply with Good Manufacturing Practice regulations to produce cell line ND50039. Thus, the pluripotent stem cells preferably meet the requirements of Good Manufacturing Practice.

[0045] A cell or pluripotent stem cell that is "lacking endogenous MHC class I molecules presented on the cell surface" does not present functional MHC class I molecules on its surface, i.e., on the surface of the cell or pluripotent stem cell, and does not contain functional MHC class I molecules in its cell membrane. In this context, the term "endogenous" refers to any MHC class protein I that is naturally contained in the cell or pluripotent stem cell and has not been artificially introduced. However, this increases the risk of rejection by the recipient's immune system, because the lack of MHC class I molecules on the cell surface can be interpreted by the immune system as a "loss of self" signal. Thus, the limitation that the cell or pluripotent stem cell is lacking MHC class I molecules on their surface does not apply to immunomodulatory proteins and / or recombinant immunomodulatory proteins that can be introduced into the pluripotent stem cell. In one embodiment, the lack of MHC class I molecules on the cell surface can be achieved by disrupting all copies of the β2-microglobulin gene in the pluripotent stem cell. The MHC complex is a heterodimer of α-microglobulin and β2-microglobulin. Thus, if β2-microglobulin is missing, functional MHC class I complexes cannot be assembled and, consequently, MHC class I molecules are not present on the cell membrane and / or cell surface.

[0046] Many possible methods are known to those skilled in the art for modifying the genome of pluripotent stem cells so that they lack MHC class I molecules and contain immunomodulatory proteins. It should be noted that the pluripotent stem cells of the present invention that lack MHC class I molecules can express immunomodulatory proteins, even if they are MHC class I molecules such as HLA-E as described herein. Thus, the term "lacking MHC class I molecules" can refer to endogenous MHC class I molecules and does not exclude the presence of (recombinant) immunomodulatory proteins.

[0047] In the pluripotent stem cell of the present invention, the B2M gene can be disrupted, so that functional endogenous B2M protein is not produced from the disrupted locus.In some embodiments, the disruption causes the expression of non-functional B2M protein, including but not limited to truncation, deletion, point mutation and insertion.In other embodiments, the disruption does not cause protein expression from the B2M gene.

[0048] Pluripotent stem cells that lack B2M expression cannot express HLA class I protein on cell surface.HLA class I deficiency provides additional advantages; for example, cells that do not have HLA class I expression cannot present self-antigens, which would otherwise prevent successful cell therapy for autoimmune diseases such as diabetes and rheumatoid arthritis.Similarly, the therapeutic gene product (e.g., dystrophin) that is introduced by the cell therapy of the present invention, which is missing in patients with certain genetic diseases (e.g., muscular dystrophy), will not be presented and recognized by the immune system as neoantigens in replacement therapy.

[0049] Any suitable technique for disrupting one, two or all copies of the B2M gene may be used; exemplary techniques are disclosed throughout this application and are within the level of ordinary skill in the art based on the teachings herein and known in the art. Exemplary other techniques may be found, for example, in U.S. Patent Application Publication No. US 2008 / 0219956, published September 11, 2008. These techniques may optionally include removing non-human DNA sequences from cells after B2M gene disruption.

[0050] An exemplary embodiment of this method is to use an adeno-associated virus (AAV) gene targeting vector, optionally including removing the transgene used for targeting by techniques such as those described below, or removing the transgene used for targeting by Cre-mediated loxP recombination, or other suitable recombination techniques. See Khan et al. (2011), Protocol, 6:482-501. It is within the level of the skilled artisan to use various techniques to generate the B2M- / - pluripotent stem cells of the present invention, i.e., pluripotent stem cells that lack MHC class I molecules, preferably human cells, based on the teachings herein and known in the art.

[0051] Disruption of B2M and / or insertion of genes encoding immunomodulatory proteins can also be performed by using engineered nucleases. These nucleases can introduce single-strand and / or double-strand breaks in DNA. The engineered nucleases can be selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9).

[0052] CRISPR describes a family of DNA sequences in bacteria. The sequences contain fragments of DNA from viruses that have attacked the bacteria. These fragments are used by the bacteria to detect and destroy DNA from similar viruses during subsequent attacks. These sequences play a key role in the bacterial defense system and form the basis of a technology known as CRISPR / Cas9 that effectively and specifically alters genes within organisms. The CRISPR / Cas system is originally a prokaryotic immune system that confers resistance to foreign genetic elements such as those present in plasmids and phages, providing a form of adaptive immunity. RNA harboring spacer sequences helps Cas (CRISPR-associated) proteins to recognize and cleave exogenous DNA. Other RNA-guided Cas proteins cleave foreign RNA. A simple version of the CRISPR / Cas system, CRISPR / Cas9, has been engineered to edit genomes. By delivering Cas9 nuclease complexed with synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at desired locations, allowing existing genes to be removed and / or new genes to be added.

[0053] There is also a CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small RNA molecules named CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). It has been shown that the Cas9 from the S. thermophilus CRISPR system can be reprogrammed to target their chosen sites by modifying the sequence of its crRNA. Thus, the present invention also relates to a method of the present invention, in which the disruption of B2M and / or the insertion of an immunomodulatory protein is mediated by an engineered nuclease, the engineered nuclease being CRISPR / Cas9. In this method, the crRNA is TIFF2025038086000003.tif19167. The Cas9 endonuclease of S. pyogenes requires a protospacer adjacent motif (PAM) in the crRNA 3' to the recognition sequence to enable binding to this specified DNA sequence. This PAM has the consensus sequence XGG, where X can be any nucleic acid. Thus, SEQ ID NOs: 2-4 may further have the sequence XGG at the 3' prime end, where X can be any nucleic acid. Thus, the crRNA may also It could be TIFF2025038086000004.tif22161.

[0054] It is within the ability of a person skilled in the art to determine whether a pluripotent stem cell lacks MHC class I molecules. Analysis can be performed at the genomic, transcriptional and / or translational levels. For example, if the lack of MHC class I molecules is achieved by destroying a gene that is essential for forming MHC class I molecules, such as the B2M gene, with a nuclease such as CRIPSR, the mutation that prevents the expression of functional B2M protein in the B2M gene can be analyzed by sequencing the respective nucleotide sequences. Examples of sequencing techniques include Sanger sequencing, as well as next-generation sequencing, such as single molecule real-time sequencing (Pacific Biosciences), ion semiconductor (Ion Torrent sequencing), pyrosequencing, sequencing by synthesis (Illumina), sequencing by ligation (SOLiD sequencing) and nanopore sequencing. Alternatively, PCR or Southern blot analysis can be performed with primer binding in the region to be mutated. The transcription of the gene encoding at least a part of MHC class I molecule can be analyzed, for example, by quantitative PCR using a primer pair that spans the region to be mutated.Finally, protein expression or translation analysis can be performed.The exemplary method for analyzing whether pluripotent stem cells lack MHC class I molecule can therefore include immunoassays, such as Western blot, flow cytometry, surface plasmon resonance, etc.

[0055] The present invention also relates to a nucleic acid comprising at least one of the sequences of SEQ ID NO: 2-7. In one embodiment, the nucleic acid may be operably linked to an expression control sequence that allows overexpression of the nucleic acid in a host cell. Exemplary expression control sequences include a promoter, such as a U6 promoter or a CMV promoter. The nucleic acid of the present invention may also be included in a vector. The vector may further encode a transactivating crRNa (tracrNA) and / or a Cas9 nuclease. The present invention also relates to the use of the nucleic acid or vector of the present invention to disrupt B2M.

[0056] The term "non-immunogenic" as used herein refers to tissue that does not essentially induce an immune response, i.e., tissue that is not rejected by the recipient. Additional characteristics of non-immunogenic engineered tissues may include that the tissue is not recognized as allogeneic by effector T cells, and / or does not bind anti-HLA antibodies, and / or is resistant to NK-mediated (natural killer cell) lysis. Assays for examining these characteristics are well known to those skilled in the art and are exemplified in Gornalusse et al. (2017), Nature Biotechnology, 35(8):765-772 and WO 2012 / 145384.

[0057] Recognition of tissue as allogeneic by effector T cells and resistance to NK-mediated lysis can be analyzed, for example, by performing a chromium release assay using NK cells. Such an assay can be performed by: 51 Exposure of the analyzed tissue or cells to Cr, CD8 + Based on subsequent contact of the tissue or cells with T cells or NK cells and final measurement of radioactivity in the supernatant by scintillation (see again, e.g., Gornalusse et al (2017), Nature Biotechnology, 35(8):765-772 and WO 2012 / 145384).

[0058] The term "effector T cells", as used herein, refers to various T cell types that immediately and actively respond to stimuli such as costimulation. It includes helper T cells and killer T cells. Helper T cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells also express the CD4 glycoprotein on their surface, and therefore are known to be CD4 +They are also known as T cells. Helper T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support an active immune response. Cytotoxic T cells (TC cells, CTL, T-killer cells, killer T cells) destroy virus-infected or tumor cells and are also involved in graft rejection. These cells also express the CD8 glycoprotein on their surface and are therefore known as CD8 + Also known as T cells. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, which prevents autoimmune disease.

[0059] The binding of anti-HLA antibodies, preferably anti-HLA-A or anti-HLA-B antibodies, to tissues of the present invention is related to complement-dependent cytotoxicity (CDC). Cells expressing HLA-A or HLA-B molecules are prone to detection by anti-HLA-A or anti-HLA-B. In an environment containing all components of the complement system and anti-HLA-A and / or anti-HLA-B antibodies, tissues expressing HLA-A and / or HLA-B on their surface are killed by the complement system if their HLA is incompatible with that of the recipient. Such tissues are likely to be rejected in the recipient. An example of such an assay can again be found in Gornalusse et al. (2017), Nature Biotechnology, 35(8):765-772.

[0060] "Immunomodulating protein" as described herein refers to any protein that can prevent targeted immune response against engineered tissue. In the context of missing functional B2M on cell surface, the cells of engineered tissue do not present any MHC I molecules on their surface, which is a "loss of self" signal that leads to their destruction by the recipient's immune system, mainly due to the action of natural killer cells. Suitable immune modulating genes include, but are not limited to, genes encoding viral proteins that inhibit antigen presentation, and preferably genes encoding single chain (SC) fusion human leukocyte antigen (HLA) class I proteins as described herein. The immune modulating proteins of the present invention may be recombinant and / or may not naturally occur in pluripotent stem cells. Thus, pluripotent stem cells may express recombinant immune modulating proteins.

[0061] The fusion protein of the SC-HLA class I fusion protein used in the present invention can be expressed by pluripotent stem cells and / or cells forming engineered tissues. The advantage of the fusion protein is that a functional HLA class I protein can be presented on the cell surface of the cells of the engineered tissue without the need to express B2M, which further associates with other HLA monomers and then induces rejection again. While the cells used in the present invention lack the expression of functional B2M, such SC-HLA class I fusion proteins can include a portion of B2M covalently linked to at least a portion of a selected group of HLA class I α chains consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G, according to the disclosure of Gornalusse et al. (2017), Nature Biotechnology, 35(8):765-772 (see, for example, FIG. 1 therein). In one embodiment, the single chain fusion HLA class I protein comprises (at least) a portion of B2M and at least a portion of HLA-A. In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201 (see also WO 2012 / 145384 in this respect). In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E. In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G. In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B. In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C. In one embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F. In one embodiment, the immunomodulatory protein is a fusion protein of B2M and HLA-E and / or comprises SEQ ID NO: 18. A vector for expression of a fusion protein of B2M and HLA-E may comprise SEQ ID NO: 17.

[0062] The pluripotent stem cells may further express a target peptide antigen presented by a single-chain fusion HLA class I protein on the cell surface. Such a target peptide antigen may stabilize the HLA class I protein and further enhance the "self" signal. In certain embodiments, the target peptide antigen is covalently linked to the single-chain fusion HLA class I protein. The HLA-E single-chain dimer may consist of an HLA-E heavy chain covalently fused to B2M through a flexible (G4S)4 linker, so that it can bind to a regular repertoire of peptides for antigen presentation (see, for example, Figure 1C in Gornalusse et al. (2017), Nature Biotechnology, 35(8):765-772). Such an HLA-E dimer is exemplified in SEQ ID NO: 18. The "HLA-E dimer" may bind to a different or "regular" peptide for antigen presentation. However, in addition or instead, a peptide may be fused to the HLA-E dimer. As an illustrative example, the HLA-E single chain trimer may contain an additional (G4S)3 linker fused to a peptide comprising the sequence VMAPRTLFL (SEQ ID NO: 1), derived from the signal sequence of HLA-G (another HLA class I molecule), a non-polymorphic peptide normally presented by HLA-E that inhibits NK cell-dependent lysis through its binding to CD94 / NGK2A. In one embodiment, the immunomodulatory protein is a fusion protein of the target peptide antigen VMAPRTLFL (SEQ ID NO: 1) fused via a (G4S)3 linker to a fusion protein of B2M and HLA-E and / or comprises SEQ ID NO: 20.

[0063] For CRISPR-mediated knock-in of gene sequences, the homologous recombination DNA repair system of the host cell can be utilized. Here, the nucleic acid containing the sequence to be inserted is flanked by "homology arms" and can be introduced into the host cell simultaneously with CRISPR / Cas9 nuclease and crRNA. The nuclease can then induce a double-strand break, which can be repaired by the DNA repair system of the host cell. In the case of homologous recombination, the DNA repair system uses a homologous sequence, usually the second allele. By introducing a nucleic acid containing a homology arm, the DNA repair system of the host cell uses the introduced nucleic acid as a template instead of the second genomic allele, thereby integrating the sequence to be inserted. "Homology arms" as used herein refer to a nucleic acid sequence whose DNA sequence is identical to the target genomic sequence. Typically, the gene sequence to be inserted is flanked by one homology arm each at the 3' and 5' ends. The "left" homology arm, i.e., the homology arm 3' of the sequence to be inserted, can have a sequence as shown in SEQ ID NO: 22 or 23. The "right" homology arm, i.e., the homology arm 5' of the sequence to be inserted, can have a sequence as shown in SEQ ID NO: 24. These exemplary homology arms mediate integration into the human B2M gene (see also FIG. 5A).

[0064] The inserted gene sequence, which may be flanked by one homology arm on each of the 3' and 5' ends and encodes an HLA-E dimer or trimer, may contain additional elements that may facilitate expression and / or function, including, but not limited to, a T2A self-cleaving peptide, e.g., as depicted in SEQ ID NO: 25, a B2M targeting signal, e.g., as depicted in SEQ ID NO: 26, or a pBHGA element (SEQ ID NO: 27).

[0065] An exemplary vector for integration of an HLA-E dimer may include SEQ ID NO: 17 (comprising a fusion protein of B2M and HLA-E), and an exemplary vector for integration of an HLA-E trimer (comprising a fusion protein of a target peptide antigen, a (G4S)3 linker, B2M and HLA-E) may include SEQ ID NO: 19. Both SEQ ID NOs: 17 and 19 include homology arms that direct integration into the B2M gene, which is knocked out as described herein. All of the exemplary vectors for integration include all of the additional elements as described herein.

[0066] Various tissues can be produced by the method of the present invention. Examples include, but are not limited to, cardiac tissue, liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue or vascular tissue. Exemplary methods for differentiating pluripotent stem cells into cell types that are essential for the function of engineered tissue, under conditions that also allow the formation of engineered tissue, can be found in the Examples section below. The differentiation of pluripotent stem cells and the formation of engineered tissue can occur simultaneously, or differentiation can be performed before formation begins.

[0067] Different approaches to producing engineered tissues are known to those skilled in the art. The following techniques can, for example, be used in the present invention: 1) tissue engineering: cells differentiated from pluripotent stem cells are mixed in a defined ratio in a hydrogel environment, see, for example, Tiburcy et al. (2017), Circulation, 135:1832-1847; 2) organoid technology: the starting material is typically undifferentiated pluripotent stem cells (HES, iPSC or PaSC), which are aggregated into so-called microtissues (see Ewart et al. (2018), Annu Rev Pharmacol Toxicol, 58:65-82), which in principle can be further fused into macrotissues or embedded in a hydrogel / matrix environment (see Lancaster et al. 2013, Nature, 501:373-379 and WO2015 / 040142); 3) 3D printing: 3D printing of tissues, ultimately similar to casting techniques (Sudo (2014), Organogenesis, 10(2):216-224; see also Tiburcy et al. (2017), Circulation, 135:1832-1847);4) Recellularization of decellularized organs: the concept is to use pig organs to recellularize for human use (for the heart see also Ott et al. (2008), Nature medicine, 14(2):213-221, but the concept is being attempted for many other organs);5) Cell sheet technology: basically, several monolayers of cells are stacked to form an organ (see, for example, Shimizu et al. (2002), Circ Res 90:e40-e48; Sawa et al. (2015), Circ J 79 :991-999).

[0068] Taking the production of functional cardiac tissue as a first illustrative example, International Patent Application WO 2015 / 040142 (herein referred to as "Bioengineered Cardiac Muscle") discloses a method for producing engineered cardiac tissue from pluripotent stem cells. The conditions described in WO 2015 / 040142 are therefore conditions for obtaining pluripotent stem cells which have been differentiated into said at least one cell type that is essential for the function of the engineered tissue, with respect to the formation of functional cardiac tissue. These conditions described in WO 2015 / 040142 may also be considered as conditions for the differentiation of pluripotent stem cells into cell types that are essential for the function of the engineered tissue, which also allow the formation of the engineered tissue, with respect to the formation of functional cardiac tissue. Thus, for the formation of engineered cardiac tissue, the method of the present invention may comprise the steps of: (i) injecting pluripotent stem cells into a culture medium containing effective amounts of (a) BMP4, activin A, FGF2, a GSK3 inhibitor, and (b) 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of an inhibitor of the Wnt signaling pathway and a serum-free supplement as described in (i), thereby inducing cardiac differentiation of the cells; and (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of a serum-free supplement as described in (i), under mechanical stimulation, thereby promoting cardiac maturation. In this context, see also Example 2 in the Examples section of this application.Other publications relating to the generation of cardiac tissue and providing conditions for differentiation of pluripotent stem cells into cell types essential for the function of the engineered tissue, optionally allowing the formation of engineered (cardiac) tissue, include, but are not limited to, Ogle et al. (2016), Sci Trans Med, 8(342), 1-7; Tiburcy et al. (2017), Circulation, 135:1832-1847; Ye et al. (2013), Cir Res, 113:922-932; Zimmermann (2009), Antioxidant & Redox Signaling, 11(8):2011-2023; Ott et al. (2008), Nature Medicine, 14(2):213-221; or Shimizu et al. (2002), 90:e40-e48. The differentiation of pluripotent stem cells and the formation of engineered tissue can occur simultaneously, or differentiation can occur before formation begins. Cardiomyocytes obtained by the method of WO 2015 / 040142 can then be processed by the method of producing engineered cardiac tissue (also known as engineered myocardium (EHM)) described in International Patent Application WO 2015 / 025030.Accordingly, the method of the invention further comprises the step of (iv) providing a serum-free reconstitution mixture in one or more forms, the reconstitution mixture comprising: (a) serum-free minimal essential medium; (b) 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml L-carnitine HCl, 0.1-10 μg / ml hydrocortisone, 0.05-5 μl / ml fatty acid supplement, 0.0001-0.1 μg / ml triiodo-L-thyronine (T3), and 0.2-2 μg / ml glycerol. and (c) the cells obtained in step (iii) and optionally a cell type, preferably human non-muscle cells, that forms part of the engineered tissue, derived from pluripotent stem cells, wherein 20-80% of the total cell mixture are the cells obtained in step (iii); wherein the reconstituted mixture has a pH of 7.2-7.6; (v) culturing the serum-free reconstituted mixture in said one or more molds, thereby causing the serum-free reconstituted mixture to compact for at least 15 minutes; (vi) culturing the mixture obtained in step (v) in said one or more molds in serum-free EHM culture medium until the mixture in said one or more molds has compacted to at least 50% of its original thickness, wherein the EHM culture medium contains at least one of: (a) 0.5-3 mmol / L Ca. 2+(b) a basal medium comprising (i) a serum-free supplement as defined in (b); (c) 0.5-10 mmol / L L-glutamine; (d) 0.01-1.0 mmol / L ascorbic acid; (e) 1-100 ng / ml IGF-1; and (f) 1-10 ng / ml TGFβ1; (vii) culturing the mixture obtained in step (iii) in serum-free EHM culture medium as defined in steps (iii) (a)-(f) under mechanical stretch, thereby forming a force-generating engineered cardiac tissue. This engineered cardiac tissue is also known as engineered myocardium (EHM). Alternatively, the method of WO 2015 / 040142 can be performed using the pluripotent stem cells of the present invention, where the pluripotent stem cells have previously colonized a hydrogel, such as type I collagen. This approach results in the creation of so-called bioengineered myocardium (BHM), which may be described as an organoid.

[0069] The skilled artisan also knows to produce other engineered tissues such as liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue or vascular tissue under conditions for differentiation of pluripotent stem cells into cell types essential for the function of the engineered tissue, which also optionally allows the formation of the engineered tissue. The differentiation of pluripotent stem cells and the formation of the engineered tissue can occur simultaneously, or differentiation can be performed before formation begins. Examples of such conditions for the production of liver tissue are disclosed in WO 2013 / 047639 or Sudo (2014), Organogenesis, 10(2):216-224. Examples of suitable conditions for the production of kidney tissue are disclosed in Morizane et al. (2017), Stem Cells, 35:2209-2217. Examples of suitable conditions for the production of brain tissue or nerve tissue are disclosed in Yang et al. (2011), Cell Stem Cell 9:517-525 or Lancaster et al. (2013), Nature, 501:373-379 (see also Example 4 of the present application). Examples of suitable conditions for the production of pancreatic tissue are disclosed in Pagliuca et al. (2014), Cell, 159:428-439 (see also Example 6 of the present application). Examples of suitable conditions for the production of functional skeletal muscle tissue are disclosed in Rao et al. (2018), Nature Communications, 9(126):1-12 (see also Example 5 of the present application). Examples of suitable conditions for the production of vascular tissue are disclosed, for example, in Song et al. (2018), Cell Stem Cell, 22:340-354. Finally, examples of suitable conditions for the generation of retinal tissue are described, for example, in Llonch et al, Developmental Biology 433 (2018) 132-143.

[0070] Many biological tissues do not contain only one single cell type. For example, cardiac muscle contains cardiomyocytes and a larger non-muscle cell component in cell abundance, which mainly includes fibroblasts and endothelial cells. Cardiomyocytes are the cell type that is essential for the function of the tissue, i.e. for the heart to beat, while fibroblasts provide the extracellular matrix that stabilizes the tissue, i.e. can be considered as cells that form part of the tissue. Endothelial cells and smooth muscle cells are involved in the cardiovascular system. Thus, the method of the present invention may further include a step of inducing differentiation of pluripotent stem cells into at least one second cell type, where the second cell type forms part of the engineered tissue. Cells that are essential for the function of the tissue and cells that form part of the tissue can be combined after differentiation to form the engineered tissue. Such methods are described, for example, in WO 2015 / 025030. Here, human cardiomyocytes and human non-muscle cells, e.g., fibroblasts, endothelial cells, smooth muscle cells or mesenchymal stem cells, are examples for cells that form part of the tissue, and cardiomyocytes are cells that are essential for the function of the tissue. By using the same pluripotent stem cells as a source for cells essential for the function of the tissue and for the (second) cells that form part of the tissue, the cells have the same genetic modifications, i.e., lack B2M and express immune-modulating proteins on the cell surface, making the combined engineered tissue non-immunogenic.

[0071] The present invention also relates to a method for producing non-immunogenic engineered tissue, but also to the non-immunogenic engineered tissue itself. Thus, the present invention relates to an engineered tissue comprising a cell type essential for the function of the engineered tissue, which is obtained by differentiating pluripotent stem cells into said cell type under conditions suitable for the differentiation of the pluripotent stem cells into said cell type, said pluripotent stem cells lacking MHC class I molecules and comprising immunomodulatory proteins on their surface, thereby rendering the engineered tissue non-immunogenic to recipients of the engineered tissue. The present invention also relates to engineered tissue obtainable by the method of the present invention. In one such embodiment, the present invention relates to an engineered tissue comprising pluripotent stem cells, which are lacking MHC class I molecules and comprising immunomodulatory proteins on their surface, and which are differentiated into a cell type essential for the function of the engineered tissue under conditions that also allow the formation of the engineered tissue, thereby rendering the engineered tissue non-immunogenic to recipients of the engineered tissue.

[0072] The engineered tissue may further comprise extracellular matrix material. Extracellular matrix (ECM) is a collection of extracellular molecules secreted by supporting cells that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of the ECM varies between multicellular structures; however, cell adhesion, intercellular communication, and differentiation are common functions of the ECM. Extracellular matrix includes interstitial matrix and basement membrane. Interstitial matrix is ​​present between various cells (i.e., in the intercellular spaces). A gel of polysaccharides and fibrous proteins fills the interstitial spaces and acts as a compressive cushion against stresses placed on the ECM. Basement membranes are sheet-like deposits of ECM that surround, for example, cardiomyocytes and endothelial cells for fixation within the tissue and to allow communication with the extracellular environment. Each type of tissue has a specific type of ECM: collagen fibers and bone salts make up the ECM of bone tissue; reticular fibers and ground substance make up the ECM of loose connective tissue; and plasma is the ECM of blood. The main ECM components of the heart are collagen type I and III, with additional contributions of hyaluronic acid and laminin, collagen type IV, proteoglycans, fibronectin and nidogen, the latter being an important component of the basement membrane. Collagen type I is the most abundant ECM material and is therefore the preferred material in tissue engineering and bioengineering. Thus, the extracellular matrix biomaterial is preferably collagen type I. In one embodiment, the formation of tissue is carried out in the presence of a hydrogel, preferably an extracellular matrix protein-containing hydrogel, such as a fibrin hydrogel or a collagen hydrogel, most preferably a collagen hydrogel.

[0073] ECM materials can also include materials that are not normally part of the natural ECM. Such non-natural ECM materials are preferably biocompatible, i.e., not toxic and do not provoke an immune response. Examples for such non-natural ECM materials include, but are not limited to, alginates, hydrogels, or synthetic matrices such as polylactic acid, polyglycolic acid, and polyglycerol sebacate (bio-rubber), and poly(octamethylene maleate (anhydride) citrate.

[0074] The engineered tissue may also have the same characteristics as the product of the method of the invention. Preferably, the engineered tissue is CD8 + The engineered tissue is not recognized as allogeneic by T cells, does not bind to anti-HLA antibodies, and / or is resistant to NK-mediated lysis. Such engineered tissue can replace at least a portion of the damaged tissue in a subject. Preferably, the engineered tissue does not bind to anti-HLA-A or anti-HLA-B antibodies.

[0075] The present invention further relates to pharmaceutical compositions containing the engineered tissues of the present invention. The pharmaceutical compositions may also contain materials such as buffers to stabilize the engineered tissues.

[0076] The engineered tissue or pharmaceutical composition of the present invention is useful in the treatment of various diseases. It is particularly preferred for use in the treatment of diseases characterized by tissue failure or dysfunction. However, the present invention also relates to the engineered tissue or pharmaceutical composition of the present invention for use in a method of treating a disease state. The present invention also relates to a method of treating a disease state comprising administering an effective amount of the engineered tissue or pharmaceutical composition of the present invention to a subject in need thereof.

[0077] The engineered tissue preferably does not contain any pluripotent stem cells, since pluripotent stem cells pose a risk of forming teratomas, particularly when the engineered tissue of the present invention is for therapeutic applications.

[0078] The disease state may be, for example, diabetes, autoimmune disease, cancer, infectious disease, cardiac disease such as myocardial infarction or heart failure, skeletal or joint conditions, muscular dystrophy, osteogenesis imperfecta, burns, liver failure, kidney failure, brain injury, or soft tissue injury, where the engineered tissue may replace tissue affected or destroyed by an autoimmune response, trauma, inadequate blood supply, or burns, to name just a few illustrative examples.

[0079] The present invention further relates to the use of the engineered tissues of the present invention in in vitro models for drug toxicity screening and / or as research tools. In this regard, the engineered tissues may serve as surrogates for human organs or tissues, for example, to avoid the use of animal models.

[0080] A better understanding of the present invention and its advantages may be obtained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0081] Example 1: Generation of pluripotent stem cells that lack MHC class I molecules and contain immunomodulatory proteins on their surface Knockout of β2-microglobulin (B2M) Pluripotent stem cell line 50039 was obtained from the NINDS Human Cell and Data Repository. This cell line is also available from Lonza and was characterized in Baghbaderani et al. (2015), Stem Cell Reports, 5:647-6659. Baghbaderani et al. also disclose standard conditions for maintaining this cell line. Alternatively, pluripotent cells were maintained in StemMACS™ iPS-Brew medium. For extracellular matrix provision, CTG Laminin-521 (Biolamina) or Geltrex (Thermo Scientific) were used.

[0082] To disrupt all copies of the B2M gene, Alt-R® CRISPR / Cas technology from Integrated DNA Technologies was used according to the manufacturer's instructions. Depending on the sequence of the CRISPR-Cas9 crRNA, Alt-R Cas9 nuclease can specifically introduce double-strand breaks, which can generate non-homologous end joining, which ultimately introduces mutations that disrupt the function of the gene. Three different crRNAs were used: TIFF2025038086000005.tif13154 Figure 1A shows an overview of the B2M gene surrounding exon 1 and the binding sites of the different crRNAs (underlined). All three crRNAs were designed to bind close to the region of the B2M gene that forms the N-terminus of the B2M translation product. One of these crRNAs, Alt-R Cas9 nuclease and Alt-R Cas9 electroporation enhancer were used to form a ribonucleoprotein complex that was electroporated into PSCs. For each of the different crRNAs, the crRNA:tracRNA:Cas9 RNP complex was prepared according to the manufacturer's instructions. The crRNA:tracRNA:Cas9 RNP complex was transfected into PSCs using the Lonza 4D Nucleofector System (X-unit) and the P3 Solution Kit (Lonza, V4XP-3012) using the program CB-150. After transfection, PSCs were plated and cultured until colonies appeared. Individual colonies were manually picked and passaged using standard protocols. Genome editing was confirmed by sequencing. From 60 sequenced colonies, 15 showed mutations within exon 1 of B2M.

[0083] Table 1 shows a summary of the results. Only four colonies were confirmed to be clonal (clones 3, 18, 20, and 34). As shown in Figure 2, genome editing resulted in frameshifts in one (clone 3) or two alleles (clones 18, 20, and 34).

[0084] (Table 1) Summary of sequencing results. Clones 3, 18, 20 and 34 showing successful deletion are highlighted in bold and underlined. TIFF2025038086000006.tif127147

[0085] FACS analysis of clones 3, 18, 20 and 34 and comparison with wild type iPSC cell line 50039 was performed (Figure 3). Here, the expression of B2M (APC-labeled anti-human B2M, Biolegend) and HLA-A,B,C (PE-labeled anti-human HLA-,B,C, Biolegend) was analyzed. As can be seen from Figure 3, wild type and heterozygous clone 3 shows HLA and B2M on the cell surface both unstimulated and after 24 hours of stimulation with interferon gamma. Three other clones 18, 20 and 34, containing mutations on both alleles, do not show any HLA or B2M on their cell surface even after 24 hours of stimulation with interferon gamma, thereby confirming that all clones are suitable starting cells for knock-in of HLA fusion proteins as described below.

[0086] Knock-in of HLA-E fusion protein Knock-in of HLA-E fusion proteins can be performed analogously to International Patent Application WO 2012 / 145384 starting from any of the above described clones 18, 20 and 34. Single chain B2M / HLA-E fusion proteins can be expressed in human PSCs using an integrative foamy virus vector. The foamy virus vector can contain an expression cassette with a promoter driving a B2M / HLA-E single chain fusion construct ("dimer"). The B2M / HLA-E single chain fusion protein ("dimer") can have the amino acid sequence as depicted in SEQ ID NO: 18. The vector for integration of the dimer can contain SEQ ID NO: 17. The trimeric single chain fusion construct can be expressed by expressing a covalently linked HLA-G peptide. TIFF2025038086000007.tif4128 ("trimer"). A vector for integration of the trimer may include SEQ ID NO: 19. Clones overexpressing the B2M / HLA-E fusion protein may be isolated by using flow cytometry with an antibody that binds to the fusion protein.

[0087] Such an approach may be undertaken here. B2M KO TC1133 hIPSC clone #34, in which Crispr / Cas9 targeting generated a 2 bp (CT) insertion in both alleles, causing a frameshift in gene expression, was selected as the parental B2M KO line for knocking in the HLA-E gene.

[0088] With proof of concept that the B2M gene can also be successfully targeted with B2M Crispr 2 / Cas9, the exon 1 region in B2M KO clone #34 was then targeted here with B2M Crispr 2 / Cas9 and a donor plasmid containing HLA-E-dimer (SEQ ID NO: 17) and HLAE-trimer (SEQ ID NO: 19) sequences with homology arms designed according to the modified genomic sequence of the KO strain as described (Figure 5A). Relatively higher cell death was observed after electroporation, as expected, when compared to untreated wild-type cells. However, hIPCS retained their proper morphology (Figure 5B). Transfection efficiency of over 80% was obtained, as demonstrated by flow cytometry analysis of GFP+ cells transfected in parallel with pmaxGFP plasmid (Figure 5C).

[0089] Characterization of HLA-E KI hIPSC clones hIPSCs transfected with HLA-E dimer and trimer plasmids were seeded as single cells into 96-well plates for colony formation. In addition, genomic DNA from the transfected hIPSC pool was simultaneously isolated. PCR was then performed using primers within the 5'-homology arm and donor sequence as indicated (Figure 6A). As expected, no products were detected in the parent B2M KO line, but the corresponding region was specifically amplified in the transfected pool, indicating successful gene integration in the B2M locus (Figure 6A). Given the preliminary data, up to 100 clones were screened, and approximately 50 of them were found to be positive for both upstream of HLA-E dimer (Figure 6B) and trimer integration (Figure 6C).

[0090] Clones that were potentially positive for HLA-E insertion were then genotyped. PCR amplification of the 5' to 3' homology arms was considered to verify the integration of the entire sequence (Figure 7A). Interestingly, correct amplification (HLA-E dimer: 2.6 kb and HLA-E trimer: 2.8 kb) was detected in a limited number of clones, among which HLA-E dimer clones #5 and 78 and HLA-E trimer clones #66 and 100 showed clear bands with the exclusion of the wild-type allele at 0.9 kb (Figures 7B and C).

[0091] HLA expression in HLA-E KI hIPSCs We then cultured HLA-E dimer clones #5 and 78 and HLA-E trimer clones #66 and 100 and first analyzed them for the expression of pluripotency markers (OCT4A and Nanog). All clones, including WT and B2M KO lines, were found to be more than 90% double positive for the expression of both OCT4A and Nanog, as demonstrated by flow cytometry analysis (Figure 8).

[0092] hIPSCs were then analyzed for B2M and HLA-E protein expression upon stimulation with interferon (IFN)-γ for 24 hours. WT cells expressed B2M as a positive control, while B2M KO hIPSCs were negative for B2M and HLA-E, as expected. In addition, >95% of HLA-E dimer (#5 and #78) and trimer clones (#66 and #100) were positive for B2M and HLA-E expression (Figure 9).

[0093] Example 2: Use of engineered PSCs to generate bioengineered myocardial tissue Engineered cardiomyocytes can be produced starting from PSCs using the protocols described in WO 2015 / 040142 and Tiburcy et al. (2017), Circulation, 135:1832-1847, and WO 2015 / 025030. Pluripotent stem cells from Example 1, in particular clones 18, 20 and 34, can be used in this example. The protocol includes the steps of inducing mesodermal differentiation, cardiac differentiation and cardiac maturation as described in WO 2015 / 040142, followed by the formation of directed tissue in type I collagen hydrogels as described in WO 2015 / 025030.

[0094] In a first step, PSCs must be differentiated into cardiomyocytes. This can be done, for example, as described in Tiburcy et al. (2017), Circulation, 135:1832-1847, and originally disclosed in WO 2015 / 040142. PSCs from Example 1 are plated at 5×10 in phosphate buffered saline (PBS) 1:30 on a matrigel-coated plate. 4 ~1×10 5 cells / cm 2The cells may be plated at 100 ng / mL and cultured in Knockout DMEM, 20% Knockout serum replacement, 2 mmol / L glutamine, 1% non-essential amino acids, 100 U / mL penicillin, and 100 μg / mL streptomycin (all Life Technologies) mixed 1:1 with irradiated human foreskin fibroblast (HFF) conditioned medium containing 10 ng / mL fibroblast growth factor-2 (FGF2) or TeSR-E8 (STEMCELL Technologies). After one day, the cells can be rinsed with Roswell Park Memorial Institute (RPMI) medium and then treated with RPMI, 2% B27, 200 μmol / L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 9 ng / mL activin A (R&D Systems), 5 ng / mL BMP4 (R&D Systems), 1 μmol / L CHIR99021 (Stemgent), and 5 ng / mL FGF-2 (Miltenyi Biotec) for three days. After further washing with RPMI medium, the cells can be cultured with 5 μmol / L IWP4 (Stemgent), followed by RPMI, 2% B27, 200 μmol / L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate from day 4 to day 13. Where indicated, cardiomyocytes can be metabolically purified by glucose deprivation from days 13 to 17 in glucose- and glutamine-free RPMI (Biological Industries), 2.2 mmol / L sodium lactate (Sigma-Aldrich), 100 μmol / L β-mercaptoethanol (Sigma-Aldrich), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0095] To generate the defined serum-free EHM as described in Tiburcy et al. (2017), Circulation, 135:1832-1847 and originally described in WO 2015 / 025030, cells were reconstituted in a mixture of pH-neutralized medical grade bovine collagen (LLC Collagen Solutions, 0.4 mg / EHM), concentrated serum-free medium (2x RPMI, 8% B27 (without insulin), 200 U / ml penicillin, and 200 μg / ml streptomycin), 4% B27 (without insulin), 1% non-essential amino acids, 2 mmol / l glutamine, 300 μmol / l ascorbic acid, 100 ng / ml IGF1 (AF-100-11), 10 ng / ml FGF-2 (AF-100-18B), 5 ng / ml VEGF165, and 10 ng / ml VEGF-2 (AF-100-18B). The cells can be cultured in Iscove's medium containing 5 ng / ml TGFβ1 (AF-100-20), 5 ng / ml TGFβ1 (AF-100-21C; essential during days 0-3 of culture), 100 U / ml penicillin, and 100 μg / ml streptomycin (serum-free protocol, Table 2). All growth factors were purchased from Peprotech as "Animal-free Recombinant Human Growth Factors" and transferred into a circular mold (inner / outer diameter: 2 / 4 mm; height: 5 mm). The human myocardial tissue can then be rapidly compressed within the mold and transferred, on day 3 of culture, preferably onto a flexible stretching device to promote load-induced contraction (Zimmermann et al. (2006), Nat Med, 12:452-458; Soong et al. (2012), Curr Protoc Cell Biol 55:23.8.1.-23.8.21; Tiburcy et al. (2014) Methods Mol Biol 1181:167-176; incorporated herein by reference and disclosed in WO 2007 / 054286). The medium was changed every other day. Myocardial tissue culture under stretch is performed for at least 7 days. FIG. 12 shows an exemplary result of this protocol.

[0096] Table 2. Overview of the EHM protocol TIFF2025038086000008.tif142128DMEM denotes Dulbecco's modified Eagle's medium; EHM, engineered human myocardium; FGF-2, fibroblast growth factor-2; IGF-1, insulin-like growth factor 1; RPMI, Roswell Park Memorial Institute medium; TGF-β1, transforming growth factor-β1; and VEGF 165 , vascular endothelial growth factor 165. * or other basal medium containing ≥ 1.2 mmol / L calcium.

[0097] Example 3: Use of engineered PSCs to generate bioengineered myocardial tissue hIPSCs obtained herein (see Example 1) were differentiated into cardiomyocytes (CMs) based on the protocol described in Example 2 above (Tiburcy et al. 2017 and WO 2015 / 025030). HLA-E KI hIPSC-derived CMs showed expression of sarcomeric proteins; alpha-actinin and cardiac troponin T (cTnT), with a high purity of >90% actinin+ cells (FIG. 10A).

[0098] Flow cytometry analysis revealed that WT CM expressed B2M and HLA class I molecules; HLA-B and C, but not HLA-E under basal conditions. IFN-γ treatment induced even stronger expression in B2M and HLA-B, C molecules and slightly less HLA-E expression (about 60%). As a negative control, B2M KO CM showed no HLA expression, as expected. HLA-E KI clones (both dimers and trimers) expressed B2M and HLA-E only after IFN-γ treatment and did not show any other HLA class I expression. CM differentiated from HLA-E trimer hIPSC lines showed slightly higher expression of HLA-E when compared to their counterparts in HLA-E dimers for initial analysis (HLA-E dimer CM: >85% and HLA-E trimer CM: >95% HLA-E positive; Figure 10B ).

[0099] Thus, in this example, cardiomyocytes (as the cell type essential for the function of engineered cardiac tissue) were experimentally generated from pluripotent stem cells that lack endogenous MHC class I molecules displayed on the cell surface of the pluripotent stem cells and contain immunomodulatory proteins on their surface.

[0100] This example further aims to generate non-immunogenic engineered cardiac tissue from the obtained cardiomyocytes as described herein, using the protocols of Tiburcy et al. 2017 and WO 2015 / 025030 described herein (see Example 2). Figure 11A shows a schematic diagram of the approach taken, and Figure 11B shows an image of the EHM during the manufacturing process. Figure 12 is a photograph of an exemplary EHM.

[0101] Fibroblasts are used as the second cell type that forms part of engineered tissue. These cells provide connective tissue. In this example, non-modified fibroblasts are used, i.e., fibroblasts that do not have immunogenic modification. For use in treatment, the fibroblasts that form part of engineered tissue can also be obtained from pluripotent stem cells that lack the endogenous MHC class I molecules that are presented on the cell surface of pluripotent stem cells and contain immunomodulatory proteins on their surface.

[0102] Following completion of EHM fabrication as described herein, a comparison of the force of contraction (FOC) of wild-type EHM, B2M knockout EHM, HLA-E dimer EHM, and HLA-E trimer EHM was performed (see FIG. 10C). As is evident from the results, all analyzed EHMs exhibited a Ca 2+ Increasing concentrations show increased contractile force. Interestingly, EHM derived from genetically modified PSCs exhibited higher contractile force than non-modified (WT) EHM.

[0103] In summary, this example shows that EHM (as an illustrative example of the non-immunogenic engineered tissue of the present invention) can be produced from cardiomyocytes obtained by differentiation of pluripotent stem cells into said at least one cell type, wherein the pluripotent stem cells lack endogenous MHC class I molecules presented on the cell surface of the pluripotent stem cells (B2M knockout) and contain immune-modulating proteins on their surface (HLA-E dimers / trimers).

[0104] Example 4: Use of modified PSCs to generate engineered brain tissue / neurons An example of human brain tissue production is disclosed in Lancaster et al. 2013, Nature, 501:373-379. Here, brain organoids are produced. The pluripotent stem cells of Example 1 can be used in this example.

[0105] PSCs can be maintained on CF-1-gamma-irradiated mouse embryonic stem cells (MEFs) (Global Stem) according to the WiCell protocol. On day 0 of organoid culture, PSCs less than passage 50 can be dissociated from MEFs by dispase treatment, the MEFs can be removed by gravity separation of stem cell colonies, and the PSCs can then be trypsinized to generate single cells. In total, 4,500 cells can then be plated into each well of an ultra-low binding 96-well plate (Corning) in human ES medium containing low concentration basic fibroblast growth factor (4 ng / ml) and 50 mM Rho-associated protein kinase (ROCK) inhibitor (Calbiochem). The embryoid bodies can be fed every other day for 6 days and then transferred to low attachment 24-well plates (Corning) in neural induction medium containing Dulbecco's Modified Eagle Medium (DMEM) / F12, 1:100 N2 supplement (Invitrogen), Glutamax (Invitrogen), Minimum Essential Medium-Non-Essential Amino Acids (MEM-NEAA) and 1 mg / ml heparin 50 (Sigma). The cells can then begin to form neuroepithelial tissue, which is fed every other day for 5 days. On day 11 of the protocol, the tissue can be transferred to a drop of Matrigel (BD Biosciences) by pipetting into cold Matrigel on a sheet of parafilm with a small 3 mm depression. These droplets may be allowed to gel at 37° C., after which they are removed from the parafilm and grown in differentiation medium containing a 1:1 mixture of DMEM / F12 and Neurobasal containing 1:200 N2 supplement (Invitrogen), 1:100 B27 supplement without vitamin A (Invitrogen), 3.5 μl / l 2-mercaptoethanol, 1:4,000 insulin (Sigma), 1:100 Glutamax (Invitrogen) and 1:200 MEM-NEAA. After 4 days of stationary growth, the tissue droplets may be transferred to a spinning bioreactor containing differentiation medium as above, except that B27 supplement with vitamin A (Invitrogen) may be used.Because retinoic acid has been shown to be important for neural differentiation in vivo, it can be included in the final medium used to differentiate cerebral organoids.

[0106] Example 5: Use of modified PSCs to generate engineered skeletal muscle tissue An exemplary method for inducing differentiation of PSCs into skeletal muscle tissue is disclosed in Rao et al. (2018), Nature Communications, 9(126):1-12. The pluripotent stem cells of Example 1 may be used in this example. The differentiation protocol is divided into several steps:

[0107] Myogenic differentiation of hPSCs into iMPCs PSCs can be maintained in feeder-free conditions in E8 medium (Stemcell Technologies). PSC colonies were dissociated into single cells with Accutase (Stemcell Technologies) and cultured at 1 × 10 3 / cm 2 PSCs can be maintained in E8 for expansion and then dissociated into single cells with Accutase, at a cell density of 3.3 × 104 cells / cm2 onto Matrigel (Corning) coated 6-well plates. 2 At 4 °C, cells can be seeded onto Matrigel-coated 6-well plates in E8 supplemented with Y27632 (5 μM, Tocris). The next day, E8 medium can be replaced with E6 medium and cells can be cultured for 2 days supplemented with CHIR99021 (10 μM, Selleck Chemical), after which time CHIR99021 can be removed and E6 medium supplemented with 1 μg / mL Dox (Sigma) for 18 days until induced myogenic precursor cells (iMPCs) can be sorted by FACS as described below.

[0108] Flow cytometry analysis Cells can be dissociated with 0.25% trypsin-EDTA, counted, washed with PBS, and then cultured at 2 × 10 6 ~1 × 10 7The cells can be resuspended in flow buffer at a concentration of 10 ...

[0109] iMPC Selection On day 20 of differentiation, cells can be dissociated with 0.25% trypsin-EDTA (Thermo) and washed in neutralizing medium. Separated cells can be centrifuged at 300 g for 5 minutes, then resuspended in sorting solution and filtered through a 30 μM filter (SYSMEX) to remove clusters and debris. Single cell suspensions can be kept on ice until sorting, and undifferentiated hPSCs are used as a negative control. Cells can be sorted for GFP using a MoFlo® Astrios™ cell sorter (Beckman Coulter).

[0110] iMPC Growth After sorting, iMPCs can be kept on ice in collection solution, spun down at 300 g for 5 min, resuspended in fresh E6 medium supplemented with Y27632, Dox, and bFGF, and then plated at 4 × 10 in Matrigel-coated flasks. 4 / cm 2 24-48 hours after sorting, cells can be incubated in Expansion Medium (EM) supplemented with Dox and bFGF and passaged at a 1:3 to 1:6 ratio every 3-4 days after reaching 80% confluence.

[0111] 2D differentiation of iMPCs iMPCs were plated on Matrigel-coated dishes at 1 x 10 5 / cm 2After reaching 100% confluence, the EM can be washed off with PBS and switched to differentiation medium (DM), which can be changed every other day.

[0112] Generation and differentiation of iSKM bundles Three-dimensional engineered skeletal muscle (iSKM bundles) can be formed within a polydimethylsiloxane (PDMS) mold containing two semi-cylindrical wells (7 mm long, 2 mm diameter) cast from a 3D machined Teflon master. The PDMS mold can be coated with 0.2% (w / v) Pluronic (Invitrogen) for 1 h at room temperature to prevent hydrogel adhesion. A Laser-cut Cerex® frame (9 × 9 mm) positioned around the two wells can be used to model the skeletal muscle. 2 , 1 mm wide edges) serve to anchor the bundle ends and facilitate handling and transplantation. The cell / hydrogel mixture may be injected into the PDMS well and polymerized for 30 min at 37°C. The formed iSKM bundles may be maintained on a rocking platform in EM supplemented with 1 μg / mL Dox and 1.5 mg / mL 6-aminocaproic acid (ACA, Sigma) for 4 days. The medium may then be switched to DM supplemented with 2 mg / mL ACA and 50 μg / mL ascorbic acid (Sigma), with the medium being changed daily.

[0113] Example 6: Use of modified PSCs to generate engineered pancreatic tissue An exemplary method for generating pancreatic tissue, or more specifically, insulin-secreting β-cells, is disclosed in Pagliuca et al. (2014), Cell, 159:428-439, in which pluripotent stem cells are differentiated into insulin-producing pancreatic β-cells (SC-β):

[0114] For the initiation of SC-β cell differentiation, pluripotent stem cells were cultured at 6 x 10 in mTeSR1 medium + 10 mM Y27632. 5Cells may be seeded at 1000 cells / ml. Differentiation may be initiated by changing the medium to Day Medium. Medium changes were as follows: Day 1: S1 + 100 ng / ml Activin A (R&D Systems) + 3 mM Chir99021 (Stemgent). Day 2: S1 + 100 ng / ml Activin A. Days 4, 6: S2 + 50 ng / ml KGF (Peprotech). Days 7, 8: S3 + 50 ng / ml KGF + 0.25 mM Sant1 (Sigma) + 2 mM RA (Sigma) + 200 nM LDN193189 (Day 7 only) (Sigma) + 500 nM PdBU (EMD Millipore). Days 9, 11, 13: S3 + 50 ng / ml KGF + 0.25 mM Sant1 + 100 nM RA. Days 14 and 16: S5 + 0.25 mM Sant1 + 100 nM RA + 1 mM XXI (EMD Millipore) + 10 mM Alk5i II (Axxora) + 1 mM T3 (EMD Millipore) + 20 ng / ml betacellulin (Thermo Fisher Scientific). Days 18 and 20: S5 + 25 nM RA + 1 mM XXI + 10 mM Alk5i II + 1 mM T3 + 20 ng / ml betacellulin. Days 21-35 (change every other day): S6 + 10 mM Alk5i II + 1 mM T3, thereby inducing differentiation of pluripotent stem cells into SC-β cells.

[0115] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0116] The present invention described illustratively herein may be suitably implemented in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprise", "include", "contain", etc. shall be read expansively and without limitation. In addition, the terms and expressions used herein are used as terms of description, not as terms of limitation, and there is no intention in the use of such terms and expressions to exclude the equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, although the present invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and changes of the invention embodied therein disclosed herein may be conceived by those skilled in the art, and such modifications and changes are considered to be within the scope of the present invention.

[0117] The invention is described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any member from the genus, regardless of whether the removed material is specifically described herein.

[0118] Other embodiments are within the scope of the following claims. Additionally, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0119] Sequence information SEQUENCE LISTING <110> Germeroth, Lothar <120> NON-IMMUNOGENIC ENGINEERED TISSUE AND METHODS OF PRODUCING AND USING THE SAME <150> EP 18183294.0 <151> 2018-07-1 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial <220> <223> Peptide antigen <400> 1 Click Download to save Val Met Ala Pro Arg Thr Leu Phe Leu mp3 youtube com 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> BM2 crRNA #1 <400> 2 actcacgctg purchasecctcc <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> B2M crRNA #2 <400> 3 gagtagcgcg together <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> B2M crRNA #3 <400> 4 ggccgagatg tctcgctccg <210> 5 <211> 23 <212> DNA <213> Artificial <220> <223> B2M crRNA #1 + PAM <400> 5 actcacgctg purchasecctcc agg <210> 6 <211> 23 <212> DNA <213> Artificial <220> <223> B2M crRNA #2 + PAM <400> 6 gagtagcgcg together agg <210> 7 <211> 23 <212> DNA <213> Artificial <220> <223> B2M crRNA #3 + PAM <400> 7 ggccgagatg tctcgctccg tgg <210> 8 <211> 550 <212> DNA <213> Homo sapiens <400> 8 cgcaccccag atcggagggc gccgatgtac agacagcaaa ctcacccagt ctagtgcatg ccttcttaaa catcacgaga ctctaagaaa aggaaactga aaacgggaaa gtccctctct ctaacctggc actgcgtcgc tggcttggag acaggtgacg gtccctgcgg gccttgtcct 180 gattggctgg gcacgcgttt aatataagtg gaggcgtcgc gctggcgggc attcctgaag 240 ctgacagcat tcgggccgag atgtctcgct ccgtggcctt agctgtgctc gcgctactct 300 ctctttctgg cctggaggct atccagcgtg agtctctcct accctcccgc tctggtcctt 360 cctctcccgc tctgcaccct ctgtggccct cgctgtgctc tctcgctccg tgacttccct 420 tctccaagtt ctccttggtg gcccgccgtg gggctagtcc agggctggat ctcggggaag 480 cggcggggtg gcctgggagt ggggaagggg gtgcgcaccc gggacgcgcg ctacttgccc 540 ctttcggcgg 550 <210> 9 <211> 151 <212> DNA <213> Artificial <220> <223> First allelle of clone 3 <400> 9 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggacggc 120 tatccagcgt gagtctctcc taccctcccg c 151 <210> 10 <211> 150 <212> DNA <213> Artificial <220> <223> 2nd allele of clone 3 <400> 10 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 120 atccagcgtg agtctctcct accctcccgc 150 <210> 11 <211> 137 <212> DNA <213> Artificial <220> <223> 1st allele of clone 18 <400> 11 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggtgagt 120 ctctcctacc ctcccgc 137 <210> 12 <211> 149 <212> DNA <213> Artificial <220> <223> 2nd allele of clone 18 <400> 12 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggagcta 120 tccagcgtga gtctctccta ccctcccgc 149 <210> 13 <211> 149 <212> DNA <213> Artificial <220> <223> 1st allele of clone 20 <400> 13 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggagcta 120 tccagcgtga gtctctccta ccctcccgc 149 <210> 14 <211> 149 <212> DNA <213> Artificial <220> <223> 2nd allele of clone 20 <400> 14 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggagcta 120 tccagcgtga gtctctccta ccctcccgc 149 <210> 15 <211> 152 <212> DNA <213> Artificial <220> <223> 1st allele of clone 34 <400> 15 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctctgtgc tcgcgctact ctctctttct ggcctggagg 120 ctatccagcg tgagtctctc ctaccctccc gc 152 <210> 16 <211> 152 <212> DNA <213> Artificial <220> <223> 2nd allele of clone 34 <400> 16 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60 atgtctcgct ccgtggcctt agctctgtgc tcgcgctact ctctctttct ggcctggagg 120 ctatccagcg tgagtctctc ctaccctccc gc 152 <210> 17 <211> 2338 <212> DNA <213> Artificial <220> <223> HLA-E Dimer sequence <400> 17 60. acagcaaact cacccagtct agtgcatgcc ttcttaaaca tcacgagact ctaagaaaag 120. ccctctctct aacctggcac tgcgtcgctg gcttggagac aggtgacggt ccctgcgggc cttgtcctga ttggctgggc acgcgtttaa tataagtgga 180 ggcgtcgcgc tggcgggcat tcctgaagct gacagcattc gggccgagat gtctcgctcc 240 gtggccttag ctgtgctcgc gctactctct ctttctggcc tgggagcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatgtc acgatctgtt gcgctggccg tgttggctct tctgtccctg agcggcctcg aggctatcca gcgtacgcca 420 aagattcagg tttactcacg tcatccagca gagaatgga agtcaaattt cctgaattgc tatgtgtctg ggtttcatcc atccgacatt gaagttgact tactgaagaa tggagagaga 540 attgaaaaag tggagcattc agacttgtct ttcagcaagg actggtcttt ctatctcttg tactacactg aattcacccc cactgaaaaa gatgagtatg cctgccgtgt gaccatgtg actttgtcac agcccaagat agttaagtgg gatcgcgaca tgggtggtgg cggttctggt 720 ggtggcggta gtggcggcgg aggaagcggt ggtggcggtt ccggatctca ctccttgaag 780 tatttccaca cttccgtgtc ccggcccggc cgcggggagc cccgcttcat ctctgtgggc 840 tacgtggacg acacccagtt cgtgcgcttc gacaacgacg ccgcgagtcc gaggatggtg 900 ccgcgggcgc cgtggatgga gcaggagggg tcagagtatt gggaccggga gacacggagc 960 gccagggaca ccgcacagat tttccgagtg aacctgcgga cgctgcgcgg ctactacaat 1020 cagagcgagg ccgggtctca caccctgcag tggatgcatg gctgcgagct ggggcccgac 1080 aggcgcttcc tccgcgggta tgaacagttc gcctacgacg gcaaggatta tctcaccctg 1140 aatgaggacc tgcgctcctg gaccgcggtg gacacggcgg ctcagatctc cgagcaaaag 1200 tcaaatgatg cctctgaggc ggagcaccag agagcctacc tggaagacac atgcgtggag 1260 tggctccaca aatacctgga gaaggggaag gagacgctgc ttcacctgga gcccccaaag 1320 acacacgtga ctcaccaccc catctctgac catgaggcca ccctgaggtg ctgggctctg 1380 ggcttctacc ctgcggagat cacactgacc tggcagcagg atggggaggg ccatacccag 1440 gacacggagc tcgtggagac caggcctgca ggggatggaa ccttccagaa gtgggcagct 1500 gtggtggtgc cttctggaga ggagcagaga tacacgtgcc atgtgcagca tgaggggcta 1560 cccgagcccg tcaccctgag atggaagccg gcttcccagc ccaccatccc catcgtgggc 1620 atcattgctg gcctggttct ccttggatct gtggtctctg gagctgtggt tgctgctgtg 1680 atatggagga agaagagctc aggtggaaaa ggagggagct actataaggc tgagtggagc 1740 gacagtgccc aggggtctga gtctcacagc ttgtaagcct cgactgtgcc ttctagttgc 1800 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1860 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1920 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 1980 catgctgggg ataaggctat ccagcgtgag tctctcctac cctcccgctc tggtccttcc 2040 tctcccgctc tgcaccctct gtggccctcg ctgtgctctc tcgctccgtg acttcccttc 2100 tccaagttct ccttggtggc ccgccgtggg gctagtccag ggctggatct cggggaagcg 2160 gcggggtggc ctgggagtgg ggaagggggt gcgcacccgg gacgcgcgct acttgcccct 2220 ttcggcgggg agcaggggag acctttggcc tacggcgacg ggagggtcgg gacaaagttt 2280 agggcgtcga taagcgtcag agcgccgagg ttgggggagg gtttctcttc cgctcttt 2338 <210> 18 <211> 515 <212> PRT <213> Artificial <220> <223> HLA-E Dimer protein sequence <400> 18 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 20 25 30 Val Glu Glu Asn Pro Gly Pro Met Ser Arg Ser Val Ala Leu Ala Val 35 40 45 Leu Ala Leu Leu Ser Leu Ser Gly Leu Glu Ala Ile Gln Arg Thr Pro 50 55 60 Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn 65 70 75 80 Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val 85 90 95 Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp 100 105 110 Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu 115 120 125 Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val 130 135 140 Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 165 170 175 Gly Ser Gly Ser His Ser Leu Lys Tyr Phe His Thr Ser Val Ser Arg 180 185 190 Pro Gly Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp 195 200 205 Thr Gln Phe Val Arg Phe Asp Asn Asp Ala Ala Ser Pro Arg Met Val 210 215 220 Pro Arg Ala Pro Trp Met Glu Gln Glu Gly Ser Glu Tyr Trp Asp Arg 225 230 235 240 Glu Thr Arg Ser Ala Arg Asp Thr Ala Gln Ile Phe Arg Val Asn Leu 245 250 255 Arg Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr 260 265 270 Leu Gln Trp Met His Gly Cys Glu Leu Gly Pro Asp Arg Arg Phe Leu 275 280 285 Arg Gly Tyr Glu Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Leu Thr Leu 290 295 300 Asn Glu Asp Leu Arg Ser Trp Thr Ala Val Asp Thr Ala Ala Gln Ile 305 310 315 320 Ser Glu Gln Lys Ser Asn Asp Ala Ser Glu Ala Glu His Gln Arg Ala 325 330 335 Tyr Leu Glu Asp Thr Cys Val Glu Trp Leu His Lys Tyr Leu Glu Lys 340 345 350 Gly Lys Glu Thr Leu Leu His Leu Glu Pro Pro Lys Thr His Val Thr 355 360 365 His His Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu 370 375 380 Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Gln Asp Gly Glu 385 390 395 400 Gly His Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp 405 410 415 Gly Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu 420 425 430 Gln Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Val 435 440 445 Thr Leu Arg Trp Lys Pro Ala Ser Gln Pro Thr Ile Pro Ile Val Gly 450 455 460 Ile Ile Ala Gly Leu Val Leu Leu Gly Ser Val Val Ser Gly Ala Val 465 470 475 480 Val Ala Ala Val Ile Trp Arg Lys Lys Ser Ser Gly Gly Lys Gly Gly 485 490 495 Ser Tyr Tyr Lys Ala Glu Trp Ser Asp Ser Ala Gln Gly Ser Glu Ser 500 505 510 His Ser Leu 515 <210> 19 <211> 2410 <212> DNA <213> Artificial <220> <223> HLA-E Trimer Sequence including Target Antigen Peptide <400> 19 acagcaaact cacccagtct agtgcatgcc ttcttaaaca tcacgagact ctaagaaaag 60 gaaactgaaa acgggaaagt ccctctctct aacctggcac tgcgtcgctg gcttggagac 120 aggtgacggt ccctgcgggc cttgtcctga ttggctgggc acgcgtttaa tataagtgga 180 ggcgtcgcgc tggcgggcat tcctgaagct gacagcattc gggccgagat gtctcgctcc 240 gtggccttag ctgtgctcgc gctactctct ctttctggcc tgggaagcgg agagggcaga 300 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatgtc acgatctgtt 360 gcgctggccg tgttggctct tctgtccctg agcggcctcg aggctgttat ggctccgcgg 420 actttaattt taggtggtgg cggatccggt ggtggcggtt ctggtggtgg cggctccatc 480 cagcgtacgc caaagattca ggtttactca cgtcatccag cagagaatgg aaagtcaaat 540 ttcctgaatt gctatgtgtc tgggtttcat ccatccgaca ttgaagttga cttactgaag 600 aatggagaga gaattgaaaa agtggagcat tcagacttgt ctttcagcaa ggactggtct 660 ttctatctct tgtactacac tgaattcacc cccactgaaa aagatgagta tgcctgccgt 720 gtgaaccatg tgactttgtc acagcccaag atagttaagt gggatcgcga catgggtggt 780 ggcggttctg gtggtggcgg tagtggcggc ggaggaagcg gtggtggcgg ttccggatct 840 cactccttga agtatttcca cacttccgtg tcccggcccg gccgcgggga gccccgcttc 900 atctctgtgg gctacgtgga cgacacccag ttcgtgcgct tcgacaacga cgccgcgagt 960 ccgaggatgg tgccgcgggc gccgtggatg gagcaggagg ggtcagagta ttgggaccgg 1020 gagacacgga gcgccaggga caccgcacag attttccgag tgaacctgcg gacgctgcgc 1080 ggctactaca atcagagcga ggccgggtct cacaccctgc agtggatgca tggctgcgag 1140 ctggggcccg acaggcgctt cctccgcggg tatgaacagt tcgcctacga cggcaaggat 1200 tatctcaccc tgaatgagga cctgcgctcc tggaccgcgg tggacacggc ggctcagatc 1260 tccgagcaaa agtcaaatga tgcctctgag gcggagcacc agagagccta cctggagaac 1320 acatgcgtgg agtggctcca caaatacctg gagaagggga aggagacgct gcttcacctg 1380 gagcccccaa agacacacgt gactcaccac cccatctctg accatgaggc caccctgagg 1440 tgctgggctc tgggcttcta ccctgcggag atcacactga cctggcagca ggatggggag 1500 ggccataccc aggacacgga gctcgtggag accaggcctg caggggatgg aaccttccag 1560 1620 catgaggggc tacccgagcc cgtcaccctg agatggaagc cggcttccca gcccaccatc 1680 cccatcgtgg gcatcattgc tggcctggtt ctccttggat ctgtggtctc tggagctgtg 1740 gttgctgctg tgatatggag gaagaagagc tcaggtggaa aaggagggag ctactataag 1800 gctgagtgga gcgacagtgc ccaggggtct gagtctcaca gcttgtaagc ctcgactgtg 1860 ccttttagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 1920 ggtgccactc ccactgtcct ttcctaataa aatgagaaa ttgcatcgca ttgtctgagt 1980 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaagggga ggattgggaa 2040 gaataagca ggcatgctgg ggataaggct atccagcgtg agtctctcct accctcccgc 2100 tctggtcctt cctctcccgc tctgcaccct ctgtggccct cgctgtgctc tctcgctccg 2160 tgacttccct tctccaagtt ctccttggtg gcccgccgtg gggctagtcc agggctggat 2220 ctcggggaag cggcggggtg gcctgggagt ggggaagggg gtgcgcaccc gggacgcgcg 2280 ctacttgccc ctttcggcgg ggagcagggg agacctttgg cctacggcga cgggagggtc 2340 gggacaaagt ttagggcgtc gataagcgtc agagcgccga ggttggggga gggtttctct 2400 tccgctcttt 2410 <210> 20 <211> 479 <212> PRT <213> Artificial <220> <223> HLA-E Trimer includinge target peptide antigen <400> 20 Met Ala Pro Arg Thr Leu Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln Val 20 25 30 Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys 35 40 45 Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys 50 55 60 Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser 65 70 75 80 Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr 85 90 95 Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln 100 105 110 Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser 130 135 140 His Ser Leu Lys Tyr Phe His Thr Ser Val Ser Arg Pro Gly Arg Gly 145 150 155 160 Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln Phe Val 165 170 175 Arg Phe Asp Asn Asp Ala Ala Ser Pro Arg Met Val Pro Arg Ala Pro 180 185 190 Trp Met Glu Gln Glu Gly Ser Glu Tyr Trp Asp Arg Glu Thr Arg Ser 195 200 205 Ala Arg Asp Thr Ala Gln Ile Phe Arg Val Asn Leu Arg Thr Leu Arg 210 215 220 Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln Trp Met 225 230 235 240 His Gly Cys Glu Leu Gly Pro Asp Arg Arg Phe Leu Arg Gly Tyr Glu 245 250 255 Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Leu Thr Leu Asn Glu Asp Leu 260 265 270 Arg Ser Trp Thr Ala Val Asp Thr Ala Ala Gln Ile Ser Glu Gln Lys 275 280 285 Ser Asn Asp Ala Ser Glu Ala Glu His Gln Arg Ala Tyr Leu Glu Asp 290 295 300 Thr Cys Val Glu Trp Leu His Lys Tyr Leu Glu Lys Gly Lys Glu Thr 305 310 315 320 Leu Leu His Leu Glu Pro Pro Lys Thr His Val Thr His His Pro Ile 325 330 335 Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro 340 345 350 Ala Glu Ile Thr Leu Thr Trp Gln Gln Asp Gly Glu Gly His Thr Gln 355 360 365 Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln 370 375 380 Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr 385 390 395 400 Cys His Val Gln His Glu Gly Leu Pro Glu Pro Val Thr Leu Arg Trp 405 410 415 Lys Pro Ala Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile Ala Gly 420 425 430 Leu Val Leu Leu Gly Ser Val Val Ser Gly Ala Val Val Ala Ala Val 435 440 445 Ile Trp Arg Lys Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Tyr Lys 450 455 460 Ala Glu Trp Ser Asp Ser Ala Gln Gly Ser Glu Ser His Ser Leu 465 470 475 <210> 21 <211> 29 <212> PRT <213> Artificial <220> <223> target peptide antigen <400> 21 Met Ala Pro Arg Thr Leu Phe Leu Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys 20 25 <210> 22 <211> 283 <212> DNA <213> Artificial <220> <223> 5' homology arm for integration into B2M gene <400> 22 acagcaaact cacccagtct agtgcatgcc ttcttaaaca tcacgagact ctaagaaaag 60 gaaactgaaa acgggaaagt ccctctctct aacctggcac tgcgtcgctg gcttggagac 120 aggtgacggt ccctgcgggc cttgtcctga ttggctgggc acgcgtttaa tataagtgga 180 ggcgtcgcgc tggcgggcat tcctgaagct gacagcattc gggccgagat gtctcgctcc 240 gtggccttag ctgtgctcgc gctactctct ctttctggcc tgg 283 <210> 23 <211> 285 <212> DNA <213> Artificial <220> <223> 5' homology arm for integration into B2M gene <400> 23 acagcaaact cacccagtct agtgcatgcc ttcttaaaca tcacgagact ctaagaaaag 60 gaaactgaaa acgggaaagt ccctctctct aacctggcac tgcgtcgctg gcttggagac 120 aggtgacggt ccctgcgggc cttgtcctga ttggctgggc acgcgtttaa tataagtgga 180 ggcgtcgcgc tggcgggcat tcctgaagct gacagcattc gggccgagat gtctcgctcc 240 gtggccttag ctctgtgctc gcgctactct ctctttctgg cctgg 285 <210> 24 <211> 343 <212> DNA <213> Artificial <220> <223> 3' homology arm for integration into B2M gene <400> 24 gctatccagc gtgagtctct cctaccctcc cgctctggtc cttcctctcc cgctctgcac 60 cctctgtggc cctcgctgtg ctctctcgct ccgtgacttc ccttctccaa gttctccttg 120 gtggcccgcc gtggggctag tccagggctg gatctcgggg aagcggcggg gtggcctggg 180 agtggggaag ggggtgcgca cccgggacgc gcgctacttg cccctttcgg cggggagcag 240 gggagacctt tggcctacgg cgacgggagg gtcgggacaa agtttagggc gtcgataagc 300 gtcagagcgc cgaggttggg ggagggtttc tcttccgctc ttt 343 <210> 25 <211> 21 <212> PRT <213> Artificial <220> <223> T2A self-cleaving peptide <400> 25 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 26 <211> 20 <212> PRT <213> Artificial <220> <223> B2m targeting signal <400> 26 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala 20 <210> 27 <211> 218 <212> DNA <213> Artificial <220> <223> pBHGA element <400> 27 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 60 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 120 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 180 gaggattggg aagacaatag caggcatgct ggggataa 218 <210> 28 <211> 30 <212> DNA <213> Artificial <220> <223> part of Exon 1 of B2M <220> <221> misc_feature <222> (28)..(30) <223> n is a, c, g, or t <400> 28 cctggaggct atccagcgtg agtctctnnn 30 <210> 29 <211> 60 <212> DNA <213> Artificial <220> <223> 5' homology arm <220> <221> misc_feature <222> (1)..(3) <223> n is a, c, g, or t <400> 29 nnnatgtctc gctccgtggc cttagctctg tgctcgcgct actctctctt tctggcctgg 60 <210> 30 <211> 23 <212> DNA <213> Artificial <220> <223> 3' homology arm <220> <221> misc_feature <222> (21)..(23) <223> n is a, c, g, or t <400> 30 gctatccagc gtgagtctct nnn 23

Claims

1. 1. A method for producing non-immunogenic engineered tissue from pluripotent stem cells, comprising: the pluripotent stem cells lack endogenous MHC class I molecules presented on their cell surface and contain immunomodulatory proteins on their cell surface; The method comprises: differentiating the pluripotent stem cells into at least one cell type that is essential for the function of the engineered tissue; mixing said at least one cell type in the presence of a biocompatible extracellular matrix to form said engineered tissue. and the engineered tissue is non-immunogenic to a recipient of the engineered tissue; The method.

2. the engineered tissue comprises: (a) are not recognized as allogeneic by effector T cells; and / or (b) resistant to NK-mediated lysis; 10. The method of claim 1.

3. The method of claim 1 or 2, wherein the engineered tissue does not bind to anti-HLA antibodies.

4. The method of claim 1 or 2, wherein the immunomodulatory protein is a single-chain fusion HLA class I protein.

5. The method of claim 4, wherein the single-chain fusion HLA class I protein comprises at least a portion of β2-microglobulin (B2M) covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

6. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A.

7. The method of any one of claims 4 to 6, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201.

8. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E.

9. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-G.

10. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-B.

11. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C.

12. The method of claim 4 or 5, wherein the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-F.

13. The method of any one of claims 4 to 12, wherein the pluripotent stem cells further express a target peptide antigen presented by a single-chain fusion HLA class I protein on the surface of the pluripotent cells.

14. 14. The method of claim 13, wherein the target peptide antigen is covalently linked to a single-chain fusion HLA class I protein.

15. 15. The method of claim 13 or 14, wherein the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO: 1).

16. A method described in any one of claims 1 to 15, wherein essentially all copies of the b2m gene are disrupted in the pluripotent stem cells.

17. The method of claim 16, further comprising: differentiating said pluripotent stem cells into at least one second cell type that forms a support cell type for said at least one cell type in said engineered tissue; and mixing said at least one cell type with said at least one second cell type in a defined ratio in the presence of said extracellular matrix to form said engineered tissue.

17. The method of any one of claims 1 to 16, comprising:

18. 18. The method of claim 17, wherein the second cell type is selected from the group consisting of fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, adipocytes, reticular cells, and mesenchymal stem cells.

19. 19. The method of any one of claims 1-18, wherein said engineered tissue is selected from the group consisting of cardiac tissue, liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, skeletal muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue, retinal tissue and vascular tissue.

20. The method of claim 19, wherein the manipulated tissue is brain tissue or skeletal muscle tissue.

21. The method of any one of claims 1 to 20, wherein the biocompatible extracellular matrix is ​​a fibrin hydrogel or a collagen hydrogel, most preferably a collagen hydrogel.

22. 22. The method of any one of claims 1 to 21, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells and parthenogenetic stem cells.

23. The method according to any one of claims 1 to 22, wherein said pluripotent stem cells are pluripotent stem cells of primate origin, preferably human pluripotent stem cells.

24. The method of any one of claims 1 to 23, wherein the pluripotent stem cells are induced pluripotent stem cells prepared from CD34-positive cells isolated from umbilical cord blood.

25. 25. The method of claim 24, wherein the induced pluripotent stem cells are NINDS Human Cell and Data Repository ND-50039 cells.

26. A process for further inducing differentiation of said pluripotent stem cells into at least one second cell type that forms a supporting cell type for said at least one cell type in said engineered tissue, wherein cells of said at least one cell type that is essential for the function of said engineered tissue are contacted with cells of said second cell type after differentiation to form said engineered tissue.

26. The method of any one of claims 1 to 25, further comprising:

27. 27. The method of claim 26, wherein the second cell type is selected from the group consisting of fibroblasts, endothelial cells, and mesenchymal stem cells.

28. 28. The method of any one of claims 5 to 27, wherein the disruption of B2M and / or the insertion of the immunomodulatory protein is mediated by an engineered nuclease.

29. The method described in claim 28, wherein the insertion of the immunomodulatory protein is mediated by CRISPR / Cas9 using a vector containing the nucleic acid sequence shown in SEQ ID NO: 17 or 19.

30. 30. The method of claim 28 or 29, wherein the engineered nuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9).

31. wherein the engineered nuclease is CRISPR / Cas9 and the crRNA is The method of any one of claims 28 to 30, selected from the group consisting of:

32. A method described in any one of claims 1 to 31, wherein the pluripotent stem cells are differentiated into at least one cell type while forming the engineered tissue.

33. An engineered tissue comprising a mixture comprising a cell type and a biocompatible extracellular matrix, comprising: The engineered tissue, wherein the cell type is essential for the function of the engineered tissue, and the cell type is obtained by differentiating pluripotent stem cells into the cell type under conditions suitable for differentiation of the pluripotent stem cells into the cell type, and the pluripotent stem cells lack MHC class I molecules and contain immunomodulatory proteins on their surface, thereby rendering the engineered tissue non-immunogenic to a recipient of the engineered tissue.

34. The engineered tissue of claim 33, further comprising at least one second cell type that forms a supporting cell type for a cell type essential for the function of the engineered tissue, wherein the at least one second cell type is obtained by differentiation of the pluripotent stem cells into the at least one second cell type as a cell population separate from the first cell type.

35. 33. An engineered tissue obtainable by the method of any one of claims 1 to 32.

36. 33. An engineered tissue obtained by the method of any one of claims 1 to 32.

37. The engineered tissue of any one of claims 33 to 36, selected from the group consisting of cardiac tissue, liver tissue, kidney tissue, brain tissue, pancreatic tissue, lung tissue, skeletal muscle tissue, gastrointestinal tissue, nervous tissue, skin tissue, bone tissue, bone marrow, adipose tissue, connective tissue, retinal tissue and vascular tissue.

38. The engineered tissue of claim 37, which is brain tissue or skeletal muscle tissue.

39. 39. The engineered tissue of any one of claims 33 to 38, wherein the extracellular matrix biomaterial is alginate, a hydrogel, a collagen hydrogel, a fibrin hydrogel, or a synthetic matrix such as polylactic acid, polyglycolic acid, polyglycerol sebacate (bio-rubber), or poly(octamethylene maleate (anhydride) citrate), preferably wherein the extracellular matrix biomaterial is type I collagen.

40. 40. The engineered tissue of claim 39, wherein the synthetic matrix material is polylactic acid or polyglycolic acid.

41. the engineered tissue comprises: (a) are not recognized as allogeneic by effector T cells; (b) does not bind to anti-HLA antibodies; and / or (c) resistant to NK-mediated lysis; 41. The engineered tissue of any one of claims 33 to 40.

42. 42. A pharmaceutical composition comprising the engineered tissue of any one of claims 33 to 41.

43. 43. The engineered tissue of any one of claims 33 to 41 or the pharmaceutical composition of claim 42 for treating a disease condition.

44. 44. The engineered tissue or pharmaceutical composition of claim 43, wherein said disease state is selected from the group consisting of diabetes, autoimmune disease, cancer, infectious disease, myocardial infarction, heart failure, skeletal or joint conditions, osteogenesis imperfecta, burns, liver failure, kidney failure, brain injury, and soft tissue injury.

45. (a) in in vitro models for drug toxicity screening, and / or (b) as a research tool; 42. Use of the engineered tissue according to any one of claims 33 to 41.

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