METHOD FOR GENERATING A HYPOIMMUNOGENIC PLURIPOTENT STEM CELL

Abstract

The invention provides pluripotent cells that are used therapeutically to regenerate tissues, but avoid rejection by recipients. In particular, the invention provides hypoimmunogenic pluripotent cells that avoid immune rejection by the host. The cells lack important immune antigens that trigger immune responses and are designed to avoid phagocytic endocytosis. The invention further provides universally acceptable off-the-shelf pluripotent cells and derivatives thereof for generating or regenerating specific tissues and organs.

Description

1 / 105 METHOD FOR GENERATING A PLURIPOTENT STEM CELL HYPOIMMUNOGENIC I. CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims the benefit of US Provisional Application No. 62 / 445,969, filed January 13, 2017. II. FIELD OF THE INVENTION

[002] Regenerative cell therapy is an important potential treatment for the regeneration of damaged organs and tissues. With the low availability of organs for transplantation and the long waiting time that accompanies it, the possibility of tissue regeneration by transplanting readily available cell lines into patients is understandably attractive. Regenerative cell therapy has shown promising initial results for the rehabilitation of damaged tissues after transplantation in animal models (e.g., after myocardial infarction). The propensity for the transplant recipient's immune system to reject allogeneic material, however, greatly reduces the potential effectiveness of the therapy and diminishes the possible positive effects surrounding such treatments. III. BACKGROUND OF THE INVENTION

[003] Regenerative cell therapy is an important potential treatment for the regeneration of damaged organs and tissues. With the low availability of organs for transplantation and the long waiting times that accompany it, the possibility of tissue regeneration through transplantation of readily available cell lines in patients is Petition 870260021971, dated 10 / 03 / 2026, page 10 / 25 2 / 105 understandably attractive. Regenerative cell therapy has shown promising initial results for the rehabilitation of damaged tissues after transplantation in animal models (e.g., after myocardial infarction). The propensity for the transplant recipient's immune system to reject allogeneic material, however, greatly reduces the potential effectiveness of the therapy and diminishes the possible positive effects surrounding such treatments.

[004] Autologous induced pluripotent stem cells (iPSCs) theoretically constitute an unlimited cellular source for patient-specific cell-based organ repair strategies. Their generation, however, presents technical and manufacturing challenges and is a time-consuming process that conceptually precludes any acute treatment modality. Allogeneic iPSC-based therapies are easier from a manufacturing standpoint and allow for the generation of standardized, high-quality cell products. Due to their allogeneic origin, however, such cell products would suffer rejection. By reducing or eliminating cell antigenicity, universally acceptable cell products could be produced. Because pluripotent stem cells can differentiate into any cell type of the three germ layers, the potential application of stem cell therapy is broad.Differentiation can be performed ex vivo or in vivo through the transplantation of progenitor cells that continue to differentiate and mature in the organ environment of the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the procedure and ensure that the appropriate cell population is generated before transplantation. Petition 870240080912, dated 09 / 23 / 2024, page 9 / 136 3 / 105

[005] In most cases, however, undifferentiated pluripotent stem cells are avoided in clinical transplant therapies due to their propensity to form teratomas. Conversely, such therapies tend to use differentiated cells (e.g., cardiomyocytes derived from stem cells transplanted into the myocardium of patients suffering from heart failure). The clinical applications of such pluripotent cells or tissues would benefit from a “safety feature” that controls cell growth and survival after transplantation.

[006] The technique seeks stem cells with the ability to produce cells that are used to regenerate diseased or deficient cells. Pluripotent stem cells (PSCs) can be used because they propagate and differentiate into several possible cell types. The family of PSCs includes several members generated through different techniques and having distinct immunogenic characteristics. The patient's compatibility with manipulated cells or tissues derived from PSCs determines the risk of immune rejection and the need for immunosuppression.

[007] Embryonic stem cells (ESCs) isolated from the inner cell mass of blastocysts exhibit histocompatibility antigens that are incompatible with recipients. This immunological barrier cannot be overcome by banks of human leukocyte antigen (HLA) type ESCs due to the fact that even HLA-matched PSC grafts suffer rejection due to mismatches in non-HLA molecules that function as minor antigens. To date, preclinical success of PSC-based approaches has been achieved only in immunosuppressed models or Petition 870240080912, dated 09 / 23 / 2024, page 10 / 136 4 / 105 immunodeficient, or when cells are encapsulated and protected from the host's immune system. Systemic immunosuppression used in allogeneic organ transplantation, however, is not justifiable for regenerative approaches. Immunosuppressive drugs have serious side effects and significantly increase the risk of infections and malignancies.

[008] To circumvent the problem of rejection, different techniques for generating patient-specific pluripotent stem cells have been developed. These include the transfer of a somatic cell nucleus into an enucleated oocyte (somatic cell nucleus transfer (SCNT) stem cells), the fusion of a somatic cell with an ESC (hybrid cell), and the reprogramming of somatic cells using certain transcription factors (induced PSCs or iPSCs). SCNT and iPSC stem cells, however, may have immune mismatches with the nucleus or donor cell, respectively, despite chromosomal identity. SCNT stem cells carry mitochondrial DNA (mtDNA) passed from the oocyte. Proteins encoded by mtDNA can act as relevant minor antigens and trigger rejection.DNA and mtDNA mutations and genetic instability associated with reprogramming and expansion of iPSC cultures can also create minor antigens relevant to immune rejection. This previously unknown immune obstacle decreases the likelihood of successful, large-scale manipulation of compatible patient-specific tissues using SCNT or iPSC stem cells. IV. SUMMARY OF THE INVENTION

[009] Pluripotent cells (HIP) were generated Petition 870240080912, dated 09 / 23 / 2024, page 11 / 136 5 / 105 hypoimmune cells that prevent rejection by the host's immune system. Placental syncytiotrophoblastic cells were utilized, forming the interface between maternal blood and fetal tissue. The expression of MHC I or HLA-I and MHC II or HLA-II was reduced. CD47 was increased. This pattern of impaired antigen presentation capacity and protection from innate immune clearance prevented host immune rejection. This was shown for HIP cells and particular ectoderm, mesoderm, and endoderm-derived cells into which the HIP cells were differentiated.

[010] Thus, the invention provides a method for generating a hypoimmunogenic pluripotent stem cell comprising: eliminating the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); eliminating the activity of both alleles of a CIITA gene in the iPSC; and increasing the expression of CD47 in the iPSC.

[011] In a preferred embodiment of the method, the iPSC is human, the B2M gene is human, the CIITA gene is human, and the increased CD47 expression results from the introduction of at least one copy of a human CD47 gene under the control of a promoter into the iPSC cell. In another preferred embodiment of the method, the iPSC is murine, the B2m gene is murine, the Cita gene is murine, and the increased CD47 expression results from the introduction of at least one copy of a murine CD47 gene under the control of a promoter into the iPSC cell. In a most preferred embodiment, the promoter is a constitutive promoter.

[012] In some embodiments of the methods disclosed here, the disruption in both alleles of the B2M gene results from a Clustered Regularly Interspaced Short Palindromic Repeats / Cas9 (CRISPR) reaction that disrupts both Petition 870240080912, dated 09 / 23 / 2024, p. 12 / 136 6 / 105 of the B2M gene alleles. In other embodiments of the method, the disruption in both alleles of the CIITA gene results from a CRISPR reaction that disrupts both alleles of the CIITA gene.

[013] The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more alterations that inactivate both alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of an endogenous CIITA gene; and one or more alterations causing an increased expression of a CD47 gene in the hHIP stem cell; wherein the hHIP stem cell induces a first Natural Killer (NK) cell response that is smaller than a second NK cell response induced by an induced pluripotent stem cell (iPSC) comprising said B2M and CIITA alterations, but not comprising the increased expression of the CD47 gene, and wherein the first and second NK cell responses are measured by the IFN-γ levels of NK cells incubated with the hHIP or iPSC in vitro.

[014] The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more alterations that inactivate both alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of an endogenous CIITA gene; and an alteration causing increased expression of a CD47 gene in the hHIP stem cell; wherein the hHIP stem cell induces a first T cell response in a humanized mouse strain that is inferior to a second T cell response in the humanized mouse strain induced by an iPSC, and wherein the first and second T cell responses are measured by determining the IFNγ levels of the humanized mice in an Elispot assay.

[015] The invention provides a method, including the Petition 870240080912, dated 09 / 23 / 2024, p. 13 / 136 7 / 105 HPV stem cell transplantation, which is disclosed here for a human individual. The invention further provides for the use of HHH stem cells for the preparation of a medicament for the treatment of conditions requiring cell transplantation.

[016] The invention provides a hypoimmunogenic pluripotent cell comprising an endogenous Histocompatibility Antigen Class I (HLA-I) function that is reduced when compared with a progenitor pluripotent cell; an endogenous Histocompatibility Antigen Class II (HLA-II) function that is reduced when compared with the parental pluripotent cell; and a reduced susceptibility to NK cell death when compared with the progenitor pluripotent cell; wherein the hypoimmunogenic pluripotent cell is less susceptible to rejection when transplanted into an individual as a result of the reduced HLA-I function, the reduced HLA-II function and the reduced susceptibility to NK cell death.

[017] In some embodiments, the hypoimmunogenic pluripotent cell is reduced by a reduction in the expression of the β-2 microglobulin protein. In a preferred embodiment, a gene encoding the β-2 microglobulin protein is knocked out. In a more preferred embodiment, the β-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1. In a more preferred embodiment, the β-2 microglobulin protein has the sequence SEQ ID NO: 1.

[018] In some embodiments, HLA-I function is reduced by reduced expression of the HLA-A protein. In a preferred embodiment, a gene encoding the HLA protein Petition 870240080912, dated 09 / 23 / 2024, page 14 / 136 8 / 105 A is subjected to knockout. In some modalities, HLA-I function is reduced by reducing the expression of the HLA-B protein. In a preferred modality, a gene encoding the HLA-B protein is subjected to knockout. In some modalities, HLA-I function is reduced by reducing the expression of the HLA-C protein. In a preferred modality, a gene encoding the HLA-C protein is subjected to knockout.

[019] In another embodiment, hypoimmunogenic pluripotent cells do not comprise an HLAI function.

[020] The invention provides a hypoimmunogenic pluripotent cell in which HLA-II function is reduced by a reduction in the expression of the CIITA protein. In a preferred embodiment, a gene encoding the CIITA protein is knocked out. In a more preferred embodiment, the CIITA protein has at least 90% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CIITA protein has the sequence of SEQ ID NO: 2.

[021] In some embodiments, HLA-II function is reduced by reducing the expression of the HLA-DP protein. In a preferred embodiment, a gene encoding the HLA-DP protein is knocked out. In some embodiments, HLA-II function is reduced by reducing the expression of the HLA-DR protein. In a preferred embodiment, a gene encoding the HLA-DR protein is knocked out. In some embodiments, HLA-II function is reduced by reducing the expression of the HLA-DQ protein. In a preferred embodiment, a gene encoding the HLA-DQ protein is knocked out.

[022] The invention provides hypoimmunogenic pluripotent cells that do not comprise an HLA-II function. Petition 870240080912, dated 09 / 23 / 2024, p. 15 / 136 9 / 105

[023] The invention provides hypoimmunogenic pluripotent cells with reduced susceptibility to macrophage phagocytosis or NK cell killing. The reduced susceptibility is caused by increased expression of a CD47 protein. In some embodiments, the increased CD47 expression results from a modification at a locus of the endogenous CD47 gene. In other embodiments, the increased CD47 expression results from a CD47 transgene. In a preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3.

[024] The invention provides hypoimmunogenic pluripotent cells comprising a suicide gene that is activated by a trigger that causes the hypoimmunogenic pluripotent or differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a thymidine kinase gene from herpes simplex virus (HSV-tk) and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 4. In a more preferred embodiment, the HSV-tk gene encodes a protein with the sequence with SEQ ID NO: 4.

[025] In another preferred embodiment, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC). In a more preferred embodiment, the EC-CD gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein with the sequence with SEQ ID NO: 5.

[026] In another preferred embodiment, the suicide gene encodes an inducible Caspase protein and the Petition 870240080912, dated 09 / 23 / 2024, p. 16 / 136 10 / 105 trigger is a chemical dimerization inducer (CID). In a more preferred embodiment, the inducible gene encodes the Caspase protein comprising at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes for the Caspase protein comprising the sequence SEQ ID NO: 6. In a more preferred embodiment, the CID is AP1903.

[027] The invention provides a method for producing a hypoimmunogenic pluripotent cell comprising reducing an endogenous function of Histocompatibility Antigen Class I (HLA-I) in a pluripotent cell; reducing an endogenous function of Histocompatibility Antigen Class II (HLA-II) in a pluripotent cell; and increasing the expression of a protein that reduces the susceptibility of the pluripotent cell to macrophage phagocytosis or NK cell killing.

[028] In one embodiment of the method, HLAI function is reduced by reducing the expression of a β-2 microglobulin protein. In a preferred embodiment, the expression of the β-2 microglobulin protein is reduced by eliminating a gene encoding the β-2 microglobulin protein. In a more preferred embodiment, the β-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1. In a more preferred embodiment, the β-2 microglobulin protein has the sequence SEQ ID NO: 1.

[029] In another embodiment of the method, HLA-I function is reduced by reducing the expression of the HLA-A protein. In a preferred embodiment, the expression of the HLA-A protein is reduced by knockout of a gene encoding the HLA-A protein. In another embodiment of the method, HLA-I function is reduced by reducing the expression of the HLA-B protein. In a Petition 870240080912, dated 09 / 23 / 2024, page 17 / 136 In a preferred embodiment, HLA-B protein expression is reduced by knockout of a gene encoding the HLA-B protein. In another embodiment of the method, HLA-I function is reduced by reducing HLA-C protein expression. In a preferred embodiment, HLA-C protein expression is reduced by knockout of a gene encoding the HLA-C protein.

[030] In another embodiment of the method, the hypoimmunogenic pluripotent cell does not comprise an HLA-I function.

[031] In another embodiment of the method, HLA-II function is reduced by reducing the expression of a CIITA protein. In a preferred embodiment, the expression of the CIITA protein is reduced by knocking out a gene that encodes the CIITA protein. In a more preferred embodiment, the CIITA protein has at least 90% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CIITA protein has the sequence SEQ ID NO: 2.

[032] In another embodiment of the method, HLA-II function is reduced by reducing the expression of an HLA-DP protein. In a preferred embodiment, the expression of the HLA-DP protein is reduced by knocking out a gene that encodes the HLA-DP protein. In another embodiment of the method, HLA-II function is reduced by reducing the expression of an HLA-DR protein. In a preferred embodiment, the expression of the HLA-DR protein is reduced by knocking out a gene that encodes the HLA-DR protein. In some embodiments of the method, HLA-II function is reduced by reducing the expression of an HLA-DQ protein. In a preferred embodiment, the expression of the HLA-DQ protein is reduced by knocking out a gene that encodes the HLA-DQ protein.

[033] In another form of the method, the cell Petition 870240080912, dated 09 / 23 / 2024, p. 18 / 136 12 / 105 pluripotent hypoimmunogenic does not comprise an HLA-II function.

[034] In another embodiment of the method, the increased expression of a protein that reduces the susceptibility of the pluripotent cell to macrophage phagocytosis results from a modification in an endogenous gene locus. In a preferred embodiment, the endogenous gene locus codes for the CD47 protein. In another embodiment, the increased protein expression results from the expression of a transgene. In a preferred embodiment, the transgene codes for a CD47 protein. In a more preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence SEQ ID NO: 3.

[035] Another embodiment of the method involves expressing a suicide gene that is activated by a trigger that causes the hypoimmunogenic pluripotent or differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a thymidine kinase gene from herpes simplex virus (HSV-tk) and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 4. In a more preferred embodiment, the HSV-tk gene encodes a protein with the sequence with SEQ ID NO: 4.

[036] In another embodiment of the method, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC). In a preferred embodiment, the EC-CD gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein with the sequence SEQ ID NO: 5. Petition 870240080912, dated 09 / 23 / 2024, p. 19 / 136 13 / 105

[037] In another embodiment of the method, the suicide gene encodes an inducible Caspase protein and the trigger is a specific chemical dimerization inducer (CID). In a preferred embodiment of the method, the gene encodes an inducible caspase protein comprising at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes for the inducible Caspase protein comprising the sequence with SEQ ID NO: 6. In a more preferred embodiment, the CID is AP1903. V. BRIEF DESCRIPTION OF THE DRAWINGS

[038] Figure 1A shows the rationale for the new hypoimmune pluripotent cells described herein. Fetuses are protected from rejection during pregnancy by fetomaternal tolerance. The cells have negative expression of MHC class I. They also have CD47 positively regulated. Figure 1B shows that fetomaternal tolerance is mediated by syncytiotrophoblastic cells. Figure 1C shows that syncytiotrophoblastic cells do not have MHC I and II and have elevated levels of CD47.

[039] Figure 2 shows induced murine pluripotent stem cells (miPSCs) generated from C57BL / 6 fibroblasts. Pluripotency was demonstrated by reverse transcriptase polymerase chain reaction (rtPCR). Multiple mRNAs associated with pluripotency were detected in miPSC cell extracts, but not in non-induced cells (murine parental fibroblasts).

[040] Figure 3 confirms the pluripotency of miPSC cells. C57BL / 6 miPSC cells formed teratomas in syngeneic mice, as well as nude and beige BALB / c mice. Petition 870240080912, dated 09 / 23 / 2024, p. 20 / 136 14 / 105 clear. No teratomas were formed in immunocompetent allogeneic BALB / c mice.

[041] Figure 4 shows that when β-2-microglobulin expression is eliminated in miPSC cells, MHC-I expression cannot be induced by IFN-γ stimulation (right panel). As a control, parental miPSC cells were stimulated with IFN-γ (left panel) and increased their MHC-I expression.

[042] Figure 5 shows that the miPSC^-2-microglobulin knockout additionally comprising a Ciita expression knockout (double knockout) showed no expression of baseline MHC-II and cannot be induced by TNF-α to express MHC-II.

[043] Figure 6A shows the increased expression of Cd47 from a transgene added to the double knockout of β-2-microglobulin / Ciita (iPSChipo cells). Figure 6B shows that C57BL / 6 iPSChipo cells survive in the allogeneic BALB / c environment, but the parental iPSC cells do not.

[044] Figure 7 shows one embodiment of the invention. It shows a schematic diagram of the iPSC manipulation that resulted in the hypoimmune pluripotent cells of the invention. To generate hypoimmune stem cells, first CRISPR-Cas 9 was used to eliminate both B2m alleles. Second, CRISPR-Cas 9 manipulation was used to eliminate both alleles of the Ciita gene. Third, a lentivirus was used to perform knockin of a Cd47 gene.

[045] Figure 8A schematically presents the role of B2m in the MHC I complex. A knockout of B2m depletes MHC I in mice or HLA-I in humans. Figure 8B schematically shows that Ciita is a transcription factor. Petition 870240080912, dated 09 / 23 / 2024, page 21 / 136 15 / 105 which causes MHC II expression in mice or HLA-II expression in humans. A Ciita knockout depletes MHC II or HLA-II expression.

[046] Figures 9A, 9B and 9C show that B2m- / iPSCs lack MHC-I expression, B2m- / -Ciita- / -iPSCs lack MHC-I and MHC-II and B2m- / -Ciita- / -Cd47 tg iPSCs lack MHC-I and MHC-II and overexpress Cd47.

[047] Figures 10A, 10B, 10C, 10D, and 10E show mouse models of transplanted “wild-type iPSCs” versus hypoimmune PSCs in allogeneic or syngeneic host mice. Here, the iPSCs were formed from C57BL / 6 mice, and the allogeneic mice are BALB / c. In Figure 10A, “wild-type iPSCs” formed only teratomas in the thighs of isogenic C57BL / 6 mice. In contrast, an immune response was mounted in the allogeneic host mice (BALB / c), and no teratomas grew. In Figure 10B, “wild-type iPSCs” formed only teratomas in isogenic C57BL / 6 mice. In Figure 10C, the immune response prevented teratoma formation in allogeneic BALB / c. Figure 10D compares the response of T cells (IFN-γ and IL-4) to iPSCs in syngeneic and allogeneic hosts using a point frequency assay (frequency of cells releasing IFN-γ and IL-4).The release of IFN-γ and IL-4 was very low in C57BL / 6 hosts, but increased dramatically in BALB / c. A. hosts. Figure 10E represents the responses of B cells in syngeneic and allogeneic hosts. iPSCs were incubated with serum from host animals that had previously received iPSCs. Bound immunoglobulins were measured using flow cytometry. The mean fluorescence intensity (MFI) was significantly higher in serum collected from the Petition 870240080912, dated 09 / 23 / 2024, page 22 / 136 16 / 105 allogeneic BALB / c receptor hosts.

[048] Figures 11A, 11B, 11C, 11D, and 11E show the partial effect of the B2m gene knockout on the iPSCs described above. In Figure 11A, B2m- / - iPSCs grew in isogenic C57BL / 6 mouse thighs, forming teratomas due to a lack of immune response, while a partial immune response was mounted in allogeneic host mice (BALB / c); for example, some of the transplanted cells survived. The iPSCs in Figure 11B, B2m- / - formed teratomas in syngeneic mice. In Figure 11C, partial survival (60%) was achieved in allogeneic hosts. In Figure 11D, the differences in T cell response (IFN-γ and IL-4) between the two hosts showed a mild but detectable T cell response against the B2m- / - iPSCs. Figure 11E shows the B cell responses in the different host mice, demonstrating a weaker immune response when compared to wild-type iPSCs.There was a significantly stronger immunoglobulin response after allogeneic transplantation of B2m- / - iPSCs into BALB / c compared to syngeneic transplantation into C57BL / 6. Thus, there was limited survival of B2m- / - iPSCs in allogeneic recipients.

[049] Figures 12A, 12B, 12C, 12D, and 12E show the increased partial effect of knockout of the B2m gene and the Ciita gene in iPSCs on cell survival in syngeneic and allogeneic host mice. The iPSCs in Figure 12A, B2m- / - Ciita- / - formed teratomas in the thighs of syngeneic C57BL / 6 mice due to a lack of immune response, while a partial immune response (but reduced when compared to the immune response of B2m- / -) was mounted in the mice. Petition 870240080912, dated 09 / 23 / 2024, page 23 / 136 17 / 105 allogeneic host (BALB / c). Figure 12B, B2m- / -Ciita / -iPSCs formed teratomas in the syngeneic mouse. Figure 12C shows that some cell grafts (91.7%) survive in allogeneic hosts. Figure 12D, differences in T cell responses (IFN-γ and IL-4) between the two hosts showed a slightly higher IFN-γ response in allogeneic versus syngeneic receptors. Figure 12E represents the B cell responses in the different host mice. The weaker immune response was compared with iPSCs by weight and B2m- / -iPSCs. A significant difference between allogeneic and syngeneic receptors was not observed. Overall, there was limited survival of B2m- / Ciita- / -iPSCs in allogeneic receptors that can be attributed to a measurable immune response.

[050] Figures 13A, 13B, 13C, 13D, and 13E show the effect of knockout of the B2m gene and the Ciita gene, and of performing knockout of the Cd47 transgene in iPSCs on cell survival in syngeneic and allogeneic host mice. Figure 13A, B2m- / - Ciita- / - Cd47tg iPSCs teratomas grew in the thighs of syngeneic and allogeneic C57BL / 6 hosts. All transplanted cell grafts survived. Figure 13B, B2m- / - Ciita- / - Cd47tg iPSCs formed teratomas in C57BL / 6. Figure 13C, 100% of cell grafts survived in allogeneic hosts. Figure 13D shows the lack of T cell response (IFN-γ and IL-4) in allogeneic recipients. No difference between the two hosts was observed. Figure 13E represents the lack of B cell responses to allogeneic receptors. No difference between the two hosts was observed. Thus, there was complete survival of B2m- / Ciita- / -Cd47tg iPSCs at allogeneic receptors. The same did not Petition 870240080912, dated 09 / 23 / 2024, page 24 / 136 18 / 105 were immunogenic, as they did not elicit a T cell or B cell response.

[051] Figures 14A, 14B and 14C show that the B2m- / -Ciita- / -Cd47tg iPSCs (referred to as non-immunogenic pluripotent stem cells (HIPs)) evaded the host immune system. Figure 14A, the expression of stimulatory NK cell binding did not increase in HIP cells. A fusion protein that recognizes several ligands of the NK cell transmembrane protein NKG2D was used to assess the level of activating ligands, which can activate cytolytic NK cell activity. Fusion protein binding to iPSCs is thus a general parameter for their NKG2D ligand activation expression. Figure 14B, HIP cells did not increase the expression of NK cell CD107a, a marker of NK cell functional activity. In contrast, B2m- / -Ciita- / -iPSCs induced CD107a expression in cells and thus triggered their cytolytic function. Figure 14C, IFN-γ Elispot assays with purified syngeneic NK cells from C57BL / 6 mouse spleen showed no HIP cell-induced NK cell response.Thus, NK cells were not activated to release IFN-γ. The spot frequency for HIP cells was not different from that of unstimulated NK cells (negative control). Only B2m- / - Ciita- / - iPSCs resulted in significantly increased IFN-γ spot frequencies.

[052] Figures 15A and 15B show additional data demonstrating that HIP cells avoid rejection or death by the innate immune system due to the Cd47 transgene. An in vivo NK cell assay had a mixture of 50% iPSCs and 50% HIPs injected into the NK-rich peritoneum of C57BL / 6 isogenic (syngeal) mice. Here, the Petition 870240080912, dated 09 / 23 / 2024, page 25 / 136 19 / 105 cytotoxicity is caused by NK cells. After 24 and 48 hours, peritoneal cells were recovered and separated. Figure 15A compares iPSCs with B2m- / -Ciita- / -iPSCs (without Cd47 transgene). B2m- / -Ciita- / -iPSCs were selectively killed by NK cells. Figure 15B compares iPSCs with B2m- / -Ciita / -Cd47 tg iPSCs (HIP cells). HIP cells were not selectively killed by NK cells. The 50% ratio of HIP cells among peritoneal iPSCs was maintained, indicating an absence of NK cell stimulation. Thus, while MHC-I and MHC-II knockouts made the cells highly susceptible to NK cell killing, Cd47 overexpression removed the stimulatory NK cell interaction.

[053] Figure 16 shows that the murine HIP cells of the invention exhibited a normal murine karyotype.

[054] Figures 17A, 17B, and 17C show that the murine HIP cells of the invention retained pluripotency during the manipulation process. RT-PCR analysis of generally accepted markers to indicate pluripotency is shown (Nanog, Oct 4, Sox2, Esrrb, Tbx3, Tcl1, and actin as a charge control). Pluripotent markers were expressed throughout the three-step manipulation process. Figure 17A compares iPSCs, B2m- / - iPSCs, and murine fibroblasts (negative control). B2m- / - iPSC cells retained pluripotency genes. Figure 17B shows the same analysis, but with B2m- / - Ciita- / - iPSCs. These retained the same pluripotency genes. Figure 17C shows the same analysis, but with the B2m- / - Ciita - / - Cd47 tg iPSCs (HIP cells). These cells retained the same pluripotency genes. Furthermore, histological images of teratomas that developed after transplantation of HIP cells into beige SCID mice show... Petition 870240080912, dated 09 / 23 / 2024, page 26 / 136 20 / 105 that the cell types associated with ectoderm, mesoderm, and endoderm were identified. Immunofluorescence markers for all three germ layers were detected (data not shown). Cell morphology was correct for neuroectoderm, mesoderm, and endoderm. Immunofluorescence staining for DAPI, GFAP, cytokeratin 8, and brachyuria confirmed the pluripotency of HIP cells.

[055] Figures 18A, 18B show HIP cells differentiated into mesodermal lineage cells and having lost their pluripotency markers. Figure 18A shows that pluripotent markers in HIP cells (labeled mHIP) were lost in differentiated murine endothelial cells (labeled miEC). Figure 18B shows that pluripotent markers were retained in HIP cells but not in differentiated murine smooth muscle cells (labeled miSMC). Figure 18C shows that pluripotent markers were retained in HIP cells but not in differentiated murine cardiomyocyte cells (labeled miCM). These results were confirmed by immunohistochemistry (data not shown). Endothelial cells were detected using anti-CD31 and anti-VE-cadherin antibodies, smooth muscle cells were detected using anti-SMA and anti-SM22 antibodies, and cardiomyocytes were detected with anti-Troponin I and anti-sarcomeric alpha-actinin antibodies.

[056] Figures 19A and 19B show that the cells HIP cells differentiated into endoderm lineage islet cells (iICs) that produced C-peptide and insulin. Figure 19A, differentiation markers were not detected in HIP cells, but were present in induced islet cells. Petition 870240080912, dated 09 / 23 / 2024, page 27 / 136 21 / 105 Figure 19B, induced islet cells produced insulin. Immunohistochemical staining for C-peptide confirmed these results (data not shown).

[057] Figures 20A and 20B show HIP cells differentiated into the ectodermal lineage. Figure 20A shows HIP cells in vitro and Figure 20B shows differentiated neuronal cells. Immunohistochemical staining with the neuroectodermal stem cell markers Nestin and Tuj-1 confirmed these results (data not shown).

[058] Figures 21A, 21B, and 21C show that cells differentiated from HIP cells retained the MHC I and II phenotype and Cd47 overexpression. Figure 21A compares the expression of MHC-I, MHC-II, and Cd47 between induced mouse endothelial cells (miEC) and B2m- / -Ciita- / Cd47 tg miEC cells. Figure 21B compares the expression of MHC-I, MHC-II, and Cd47 between induced mouse smooth muscle cells (miSMC) and B2m- / -Ciita- / -Cd47 tg mSMC cells. Figure 21C compares the expression of MHC-I, MHC-II, and Cd47 between induced mouse myocardocytes (miCM) and B2m- / -Ciita- / -Cd47 tg miMC cells.

[059] Figures 22A, 22B, and 22C show that endothelial cells differentiated from HIP cells are non-immunogenic. Figure 22A, transplantation of syngeneic and allogeneic C56BL / 6 miECs into BALB / c allogeneic recipient mice. miECs in BALB / c recipient mice generated a pronounced immune response, but not in syngeneic mice. This was evidenced by strong IFN-γ Elispot and immunoglobulin responses (FACS analysis) in BALB / c recipients (Figure 22B). Figure 22C, neither HIP cells nor miECs generated an immune response in syngeneic or allogeneic recipients. Petition 870240080912, dated 09 / 23 / 2024, page 28 / 136 22 / 105

[060] Figures 23A, 23B, and 23C show that induced smooth muscle cells from differentiated mice, distinct from HIP cells, are non-immunogenic. Figure 23A, transplantation of syngeneic and allogeneic C56BL / 6 miSMCs into mice. miSMCs in allogeneic BALB / c recipient mice generated a pronounced immune response, but not in syngeneic mice. This was evidenced by strong IFN-γ Elispot and immunoglobulin responses (FACS analysis) in BALB / c recipients. Figure 23C, neither HIP cells nor miSMCs generated an immune response in syngeneic or allogeneic recipients.

[061] Figures 24A, B, and C show that mouse-induced cardiomyocyte cells differentiated from HIP cells are non-immunogenic. Figure 25A, transplantation of syngeneic and allogeneic C56BL / 6 miCMCs. Figure 24B shows that miCMCs in allogeneic BALB / c recipient mice generated a pronounced immune response, but not in syngeneic mice. This was evidenced by strong IFN-γ Elispot and immunoglobulin responses (FACS analysis) in BALB / c recipients (Figure 24B). Figure 24C shows that neither HIP cells nor miCMCs generated an immune response in syngeneic or allogeneic recipients.

[062] Figure 25 shows that differentiated cells (miECS, miSMCs, miCMs) derived from HIP cells avoid rejection via the innate immune system. An NK fusion protein assay showed that none of the three differentiated cells exhibited increased expression of stimulatory NK cell ligands when compared with differentiated cells derived from miPSCs.

[063] Figures 26A and 26B show that the HIP cell-derived miECs of the invention avoided the immune reaction and achieved long-term survival in a host. Petition 870240080912, dated 09 / 23 / 2024, page 29 / 136 23 / 105 allogeneic. Figure 26A, miEC grafts derived from miPSCs showed long-term survival in syngeneic recipients (C57BL / 6), but were rejected in allogeneic recipients (BALB / c). Figure 26B, miECs derived from HIP achieve long-term survival after transplantation in syngeneic and allogeneic recipients.

[064] Figure 27: miECs derived from HIP cells organized to form vascular structures in allogeneic hosts. After transplantation within a Matrigel matrix, over six weeks, the miECs organized themselves in a three-dimensional manner to form vascular structures. These results were confirmed by immunofluorescence for luciferase and VE-cadherin; the miECs were transduced to express luciferase before transplantation. Survival was monitored by bioluminescence imaging and transplanted cells were identified with immunofluorescence staining against luciferase (data not shown).

[065] Figure 28 shows that human HIP cells exhibited a normal human karyotype.

[066] Figures 29 show that human HIP cells maintained pluripotency during the manipulation process. The hiPSCs (e.g., the starting cells, before the invention modifications) and the HIP cells of the invention have expression of the pluripotency genes (NANOG, OCT4, SOX2, DPPA4, hTERT, ZFP42, and DEMT3B; G3PDH served as a charge control) using PCR assays. Immunofluorescence staining confirmed this result, as the cells express the markers TRA-1-60, TRA-1-81, Sox2, Oct4, SSEA-4, and alkaline phosphatase (data not shown).

[067] Figures 30A and 30B show that the cells Petition 870240080912, dated 09 / 23 / 2024, page 30 / 136 24 / 105 Human HIP cells transplanted into humanized allogeneic mice did not elicit an immune response. Figure 30A shows that T cells did not respond to transplanted HIP cells, as measured by IFN-γ or IL5 production in Elispot assays. In contrast, transplanted iPSCs did. Figure 30B shows that only iPSCs elicited a strong antibody response by flow cytometry. HIP cells did not.

[068] Figures 31A, 31B, 31C, and 31D show that human HIP cells have differentiated into the mesodermal lineage. Figure 31A shows the morphology of a cell. Human HIP. Figure 31B shows HIP-derived endothelial cells stained with CD31, VE-cadherin, and DAPI as controls. Figure 31C shows HIP-derived cardiomyocytes stained with sarcomeric α-actinin, Troponin I, and DAPI as controls. Figure 31D shows premature vessel formation by HIP-derived endothelial cells. HIP-derived cardiomyocytes were observed beating (data not shown).

[069] Figures 32A and 32B show that transplanted human endothelial cells derived from human HIP cells did not elicit an immune response in allogeneic humanized mice. Figure 32A, hiECs mounted a significant T cell response in Elispot IFN-γ and IL5 assays, while hiECs derived from human HIP cells did not. Figure 32B shows the B cell response in flow cytometry. Only hiECs generated significant immunoglobulin binding, as measured by mean fluorescence intensity (MFI).

[070] Figures 33A and 33B show that the Petition 870240080912, dated 09 / 23 / 2024, page 31 / 136 25 / 105 transplantation of human cardiomyocytes derived from human HIP cells did not result in an immune response in allogeneic humanized mice. Figure 33A shows the differences in T cell responses to “wild-type” hiCMs versus B2M- / - CIITA- / - CD47tg HIP cells in IFN-γ and IL5 ELispots. Figure 33B shows the B cell response in flow cytometry. Only “wild-type” hiCMs generated a significant load of hiEC immunoglobulins, as measured by mean fluorescence intensity (MFI).

[071] Figures 34A, 34B, 34C and 34D show that the human HIP cells of the invention avoided rejection by the innate immune system. NK cells were isolated from mice. BALB / c using Magnetically Activated Cell Sorting (MACS). 5X10⁶ stimulating cells (iPSC derivatives of C57BL / 6, iEC, iSMC or iCM and B2M- / -CIITA- / - or B2M- / -CIITA / -CD47 tg) were incubated with 5X10⁶ MACS-sorted NK cells in an IFN-γ Elispot plate. After 24 hours, the spot frequency was determined with an Elispot reader. All three B2M- / -CIITA- / - derivatives induced a strong NK response. All three B2M- / -CIITA- / -CD47 derivatives, however, did not induce any NK cell response, and their spot frequency was not statistically different from the negative controls (isolated NK cells not incubated with a stimulating cell). Figure 34A shows endothelial cells. Figure 34B shows smooth muscle cells. Figure 34C shows cardiomyocytes. Figure 34D shows the positive control of YAC-1 mouse lymphoma.

[072] Figures 35A, 35B and 35C show the innate immune response (or lack thereof). A mixture of 50% by weight of derivative (5X106 cells) and 50% or Petition 870240080912, dated 09 / 23 / 2024, page 32 / 136 26 / 105 C57BL / 6 B2m- / - Ciita- / - or B2m- / - Ciita- / - Cd47 tg derivative (5X106 cells). The cells were stained with CFSE staining at 10 μM for 10 min and resuspended in 500 μL of saline solution. The cell mixture was then injected into the NK-rich peritoneum of C57BL / 6 (syngeneic) mice. In this syngeneic model, all cytotoxicity is caused by NK cells. After 48 h, peritoneal cells were recovered and their ratio was calculated. Wt and manipulated cells were identified by MHCI staining on FACS. Figure 35A shows endothelial cells. Figure 35B shows smooth muscle cells. Figure 35C shows cardiomyocytes.

[073] Figures 36A, 36B, and 36C show the genetic manipulation of human iPSCs verified by FACS. The lack of HLA I and HLA II was confirmed in B2M- / -CIITA- / hiSCs. Additionally, B2M- / -CIITA- / -CD47 tg showed high expression of CD47. Figure 36A shows the HLA I results. Figure 36B shows the HLA II results. Figure 36C shows the CD47 results.

[074] Figures 37A and B show that the immune phenotype was maintained after differentiation of B2M- / -CIITA- / -CD47 tg iPSCs. When compared with unmodified wt derivatives, FACS analysis showed that the B2M- / -CIITA- / CD47 tg derivatives lacked HLA I and HLA II and overexpressed CD47. Figure 37A shows endothelial cells and Figure 37B shows cardiomyocytes. VI. DETAILED DESCRIPTION OF THE INVENTION A. INTRODUCTION

[075] The invention provides hypoimmunogenic pluripotent cells (HIPs) that evade host immune responses due to various genetic manipulations, such as Petition 870240080912, dated 09 / 23 / 2024, p. 33 / 136 27 / 105 described here. The cells lack important immune antigens that trigger immune responses and are engineered to evade phagocytosis. This allows for the derivation of “ready-to-use” cellular products to generate specific tissues and organs. The benefit of having the ability to use allogeneic human HIP cell derivatives in human patients results in significant benefits, including the ability to avoid long-term adjunctive immunosuppressive therapy and drug use typically seen in allogeneic transplants. It also provides significant cost savings, as cell therapies can be used without the need for individual treatments for each patient. Recently, it has been shown that cellular products generated from autologous cell sources can become subject to immune rejection with few or even a single antigenic mutation. Thus, autologous cellular products are not inherently non-immunogenic.Furthermore, cell engineering and quality control are very laborious and expensive, and autologous cells are not available for acute treatment options. Only allogeneic cell products can be used for a larger patient population if the immune barrier can be overcome. HIP cells will serve as a universal cell source for the generation of universally acceptable derivatives.

[076] The present invention relates to the exploitation of fetomaternal tolerance that exists in pregnant women. Although half of the fetus's human leukocyte antigens (HLA) are paternally inherited and the fetus expresses HAP antigens with major disparities, the maternal immune system does not recognize the fetus as an allogeneic entity and does not initiate an immune response, for example, as is seen in a type of reaction Petition 870240080912, dated 09 / 23 / 2024, page 34 / 136 28 / 105 immune “host versus graft”. Fetal-maternal tolerance is primarily mediated by syncytiotrophoblastic cells at the maternal-fetal interface. As shown in Figure 7, syncytiotrophoblast cells show little or no protein from major histocompatibility complexes I and II (MHC-I and MHC-II), as well as increased expression of CD47, known as the “don’t eat me” protein that suppresses phagocytic innate immune surveillance and elimination of HLA-free cells. Surprisingly, the same tolerogenic mechanisms that prevent fetal rejection during pregnancy also allow the HIP cells of the invention to escape rejection and facilitate long-term survival and engraftment of these cells after allogeneic transplantation.

[077] These results are further surprising insofar as this fetomaternal tolerance can be introduced with only three genetic modifications (compared to the starting iPSCs, e.g., hiPSCs), two reductions in activity (“knockouts” as described here) and one increase in activity (a “knockin” as described here). Generally, others skilled in the art have attempted to suppress the immunogenicity of iPSCs, but have been only partially successful; see Rong et al., Cell Stem Cell 14:121-130 (2014) and Gornalusse et al., Nature Biotech doi:10.1038 / nbt.3860).

[078] Thus, the invention provides for the generation of HIP cells from pluripotent stem cells and then their maintenance, differentiation and finally transplantation of their derivatives into patients in need of them. B. DEFINITIONS

[079] The term “pluripotent cells” refers Petition 870240080912, dated 09 / 23 / 2024, page 35 / 136 29 / 105 to cells that can self-renew and proliferate while remaining in an undifferentiated state and that can, under appropriate conditions, be induced to differentiate into specialized cell types. The term “pluripotent cells,” as used herein, encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human Embryonic Stem Cell and the HUES collection of the Howard Hughes Medical Institute (as described in Cowan, CA et al., New England J. Med. 350:13). (2004), incorporated herein by reference in its entirety.)

[080] “Pluripotent stem cells,” as used herein, have the potential to differentiate into any of the three germ layers: endoderm (e.g., stomach lining, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissues and nervous system tissues). The term “pluripotent stem cells,” as used herein, also encompasses “induced pluripotent stem cells,” or “iPSCs,” a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of progenitor cells include somatic cells that have been reprogrammed to induce an undifferentiated pluripotent phenotype by various means. Such “iPS” or “iPSC” cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. The methods for Petition 870240080912, dated 09 / 23 / 2024, page 36 / 136 30 / 105 induction of iPS cells are known in the art and are further described below. (See, for example, Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8: 381-384 (2009); each is incorporated herein by reference in its entirety.) One generation of induced pluripotent stem cells (iPSCs) is described below. As used herein, hiPSCs are human induced pluripotent stem cells and miPSCs are murine induced pluripotent stem cells.

[081] “Characteristics of pluripotent stem cells” refers to characteristics of a cell that distinguishes pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo differentiation, under appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lines from all three germ layers (endoderm, mesoderm, and ectoderm) is a characteristic of pluripotent stem cells. Expression or non-expression of certain combinations of molecular markers are also characteristics of pluripotent stem cells. For example, human pluripotent stem cells express at least several and, in some embodiments, all of the markers in the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF1, Oct4, Rex1, and Nanog. Cell morphologies associated with stem cells Pluripotent characteristics are also found in pluripotent stem cells.As described here, cells do not need to go through pluripotency to be... Petition 870240080912, dated 09 / 23 / 2024, page 37 / 136 31 / 105 reprogrammed into endodermal progenitor cells and / or hepatocytes.

[082] As used herein, multipotent or “multipotent cell” refers to a type of cell that can give rise to a limited number of other particular cell types. For example, multipotent cells have the ability to form endodermal cells. Additionally, multipotent blood stem cells can differentiate into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.

[083] As used herein, the term oligopotent refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells have the ability to form cells of lymphoid or myeloid lineages, respectively.

[084] As used herein, the term unipotent means the ability of a cell to form only one type of cell. For example, spermatogonial stem cells only have the ability to form spermatozoa.

[085] As used herein, the term totipotent means the ability of a cell to form an entire organism. For example, in mammals, only the blastomeres of the zygote and the first cleavage stage are totipotent.

[086] As used herein, non-pluripotent cells refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, without limitation, cells from a selected tissue of bone marrow, skin, skeletal muscle, Petition 870240080912, dated 09 / 23 / 2024, page 38 / 136 32 / 105 adipose tissue and blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The initial cells used to generate induced multipotent cells, endodermal progenitor cells, and hepatocytes may be non-pluripotent cells.

[087] Differentiated cells include, without limitation, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In particular embodiments, a less potent cell is considered differentiated in reference to a more potent cell.

[088] A somatic cell is a cell that forms the body of an organism. Somatic cells include cells that form organs, skin, blood, bones, and connective tissue in an organism, but not germ cells.

[089] Cells may be from, for example, human or non-human mammals. Exemplary non-human mammals include, without limitation, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, a cell is from an adult human or non-human mammal. In some embodiments, a cell is from a neonatal human, an adult human, or a non-human mammal.

[090] As used herein, the terms individual or patient refer to any animal, such as a domesticated animal, zoo animal, or a human being. The individual or patient may be a mammal such as a dog, cat, bird, cattle, or a human being. Specific examples of individuals and patients include, without Petition 870240080912, dated 09 / 23 / 2024, page 39 / 136 33 / 105 limitation, individuals (particularly humans) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow, and similar tissues.

[091] Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, without limitation, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates (e.g., chimpanzees, monkeys, and baboons).

[092] By “hypoimmunogenic pluripotent cell” or “HIP cell” is meant here a pluripotent cell that retains its pluripotent characteristics and gives rise to a reduced immune rejection response when transferred to an allogeneic host. In preferred embodiments, HIP cells do not originate an immune response. Thus, “hypoimmunogenic” refers to a significantly reduced or eliminated immune response when compared with the parental immune response cell (i.e., “wt”) before immunomanipulation, as presented here. In many cases, HIP cells are immunologically silent and still retain pluripotent capabilities. Assays for HIP characteristics are described below.

[093] The “HLA” or “human leukocyte antigen” complex is a gene complex that encodes the proteins of the major histocompatibility complex (MHC) in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two MHCs, class I and class II, “HLA-I” and “HLA-II”. HLA-I includes three proteins, Petition 870240080912, dated 09 / 23 / 2024, page 40 / 136 34 / 105 HLA-A, HLA-B, and HLA-C, which present peptides from inside the cell, and antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with β-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4+ cells (also known as helper T cells). It should be understood that the use of MHC or HLA is not intended to be limiting, as it depends on whether the genes are from humans (HLA) or murine (MHC). Thus, with regard to mammalian cells, these terms may be used interchangeably in this document.

[094] By “gene knockout” is meant here a process that renders a particular gene inactive in the host cell in which it resides, resulting in no protein of interest being produced or in an inactive form. As will be appreciated by those skilled in the art and described further on, this can be achieved in several different ways, including removing nucleic acid sequences from a gene, or disrupting the sequence with other sequences, altering the reading frame, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest may be removed or replaced with nonsense sequences; all or part of a regulatory sequence, such as a promoter, may be removed or replaced; translation initiation sequences may be removed or replaced, etc.

[095] By “gene knockin” we mean here a process that adds a genetic function to a cell. Petition 870240080912, dated 09 / 23 / 2024, p. 41 / 136 35 / 105 host. This causes increased levels of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in several ways, including adding one or more additional copies of the gene to the host cell or altering a regulatory component of the endogenous gene, increasing protein expression. This can be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[096] The protein β-microglobulin” or β2M” or B2M refers to the human β2M protein which has the amino acid and nucleic acid sequences shown below; the human gene has the accession number NC_000015.10: 44711487-44718159.

[097] The CD47 protein” refers to the human β2M protein which has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000016.10: 10866208-10941562.

[098] The CIITa protein” refers to the human CIITA protein which has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000003.12: 108043094-108094200.

[099] By “wild type” in the context of a cell means a cell found in nature. However, in the context of a pluripotent stem cell, as used herein, it also means iPSCs that may contain nucleic acid alterations resulting in pluripotency, but have not undergone the gene-editing processes of the invention to achieve hypoimmunogenicity.

[0100] By "syngeneic" here refers to the genetic similarity or identity of a host organism Petition 870240080912, dated 09 / 23 / 2024, p. 42 / 136 36 / 105 and cell transplantation where there is immunological compatibility; for example, no immune response is generated.

[0101] Allogeneic here refers to the genetic dissimilarity of a host organism and cell transplant where an immune response is generated.

[0102] By B2M- / - here it means that a diploid cell has had the B2M gene inactivated on both chromosomes. As described here, this can be accomplished in a variety of ways.

[0103] By CIITA- / - here it means that a diploid cell has had the CIITA gene inactivated on both chromosomes. As described here, this can be accomplished in a variety of ways.

[0104] By CD47 tg (represented by transgene) or CD47+) it is understood that this host cell expresses CD47, in some cases having at least one additional copy of the CD47 gene.

[0105] An Oct polypeptide refers to any of the Octamer families of naturally occurring transcription factors, or variants thereof that retain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the nearest naturally occurring family member, or polypeptides that comprise at least the DNA-binding domain of the naturally occurring family member, and may further comprise a transcription activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3 / 4 (herein referred to as Oct4) contains the POU domain, a conserved 150-amino acid sequence between Pit-1, Oct-1, Oct-2, and uric-86. Petition 870240080912, dated 09 / 23 / 2024, page 43 / 136 37 / 105 (See, Ryan, AK & Rosenfeld, MG, Genes Dev. 11:12071225 (1997), incorporated herein by reference in its entirety.) In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity in their complete sequence compared to a naturally occurring Oct polypeptide family member, such as those listed above or as listed in Genbank accession number NP002692.2) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3 / 4 or Oct4) may be from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used with the manipulated cell species. The Oct polypeptide (or polypeptides) may be a pluripotency factor that can help induce multipotency in non-pluripotent cells.

[0106] A Klf polypeptide refers to any of the naturally occurring members of the Krüppel-like factor (Klf) family, zinc finger proteins containing amino acid sequences similar to those of the Drosophila Krüppel embryonic pattern regulator, or variants of the naturally occurring members that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and may further comprise a transcriptional activation domain. (See Dang, DT, Pevsner, J. & Yang, VW, Cell Biol. 32:1103-1121 (2000), incorporated herein by reference in its entirety.) Exemplary members of the Klf family include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Petition 870240080912, dated 09 / 23 / 2024, p. 44 / 136 38 / 105 Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factors capable of generating iPS cells, as were the related genes Klf1 and Klf5, although with reduced efficiency. (See Nakagawa et al., Nature Biotechnology 26:101-106 (2007), incorporated herein by reference in its entirety.) In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity in their complete sequence compared to a naturally occurring Klf polypeptide family member, such as those listed above or as listed in Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used with the manipulated cell species. The Klf polypeptide (or polypeptides) can be a pluripotency factor.The expression of the Klf4 gene or polypeptide can help induce multipotency in a single starter cell or in a population of starter cells.

[0107] A “Myc polypeptide” refers to any of the naturally occurring members of the Myc family. (See, for example, Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6:635-645 (2005), incorporated herein by reference in its entirety.) It also includes variants that maintain transcription factor-like activity when compared to the closest naturally occurring family member (i.e., with at least 50%, 80%, or 90% activity). Additionally, it includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member, and may further comprise a Petition 870240080912, dated 09 / 23 / 2024, page 45 / 136 39 / 105 transcriptional activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity in their complete sequence compared to a naturally occurring Myc polypeptide family member, such as those listed above or as listed in Genbank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) may be from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used with the manipulated cell species. The Myc polypeptide (or polypeptides) may be a pluripotency factor.

[0108] A “Sox polypeptide” refers to any of the naturally occurring members of the SRY-related HMG-box (Sox) transcription factors characterized by the presence of the high mobility group (HMG) domain, or variants thereof, that maintain similar transcription factor activity when compared with the closest related natural family member (i.e., with at least 50%, 80%, or 90% activity). In addition, it includes polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and may additionally comprise a transcriptional activation domain. (See, for example, Dang, DT et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000), incorporated herein by reference in its entirety.) Exemplary Sox polypeptides include, for example, Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30.It has been shown that Sox1 produces iPS cells with similar efficiency to Sox2, and. Petition 870240080912, dated 09 / 23 / 2024, p. 46 / 136 40 / 105 The Sox3, Sox15, and Sox18 genes have also been shown to generate iPS cells, although with slightly less efficiency than Sox2. (See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007), incorporated herein by reference in its entirety.) In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity in their complete sequence compared to a naturally occurring member of the Sox polypeptide family such as those listed above or listed in Genbank accession number CAA83435 (human Sox2). Sox polypeptides (e.g., Soxl, Sox2, Sox3, Sox15, or Sox18) may be from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used with the manipulated cell species. The polypeptide (or polypeptides) Sox can be a pluripotency factor. As discussed here, SOX2 proteins find particular use in the generation of iPSCs.

[0109] By “differentiated hypoimmunogenic pluripotent cells” or “differentiated HIP cells” or “dHIP cells” is meant here iPS cells that have been manipulated to have hypoimmunogenicity (for example, through the elimination of B2M and CIITA and the inactivation of CD47) and then they are differentiated into a cell type for definitive transplantation into individuals. Thus, for example, HIP cells can be differentiated into hepatocytes (“dHIP hepatocytes”), into pancreatic beta cells or islet organoids (“dHIP beta cells”), into endothelial cells (“dHIP endothelial cells”), etc.

[0110] The term percentage of “identity”, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or Petition 870240080912, dated 09 / 23 / 2024, page 47 / 136 41 / 105 subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum match, measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percentage of identity may exist over a region of the sequence to be compared, for example, over a functional domain, or alternatively, it may exist along the entire length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence against which the tests are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and the sequence algorithm program parameters are assigned.The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence (or sequences) relative to the reference sequence, based on the designated program parameters.

[0111] Optimal sequence alignment for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Petition 870240080912, dated 09 / 23 / 2024, page 48 / 136 42 / 105 Madison, Wis.), or by visual inspection (see generally Ausubel et al., below).

[0112] An example of an algorithm that is stable for determining sequence identity percentage and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0113] Inhibitors, activators, and modulators affect the function or expression of a biologically relevant molecule. The term modulator includes both inhibitors and activators. They can be identified using in vitro and in vivo assays for the expression or activity of a target molecule.

[0114] Inhibitors are agents that, for example, inhibit expression or bind to target molecules or proteins. They may partially or completely block stimulation or have protease inhibitory activity. They may reduce, decrease, prevent, or delay activation, including inactivation, desensitization, or downregulation of the activity of the described target protein. Modulators may be antagonists of the target molecule or protein.

[0115] Activators are agents that, for example, induce or activate the function or expression of a target molecule or protein. They can bind to, stimulate, increase, open, activate, or facilitate the activity of the target molecule. Activators can be agonists of the target molecule or protein.

[0116] Homologues are bioactive molecules that are similar to a reference molecule in nucleotide sequence, peptide sequence, functional level or Petition 870240080912, dated 09 / 23 / 2024, p. 49 / 136 43 / 105 structural. Homologs may include derivatives of sequences that share a certain percentage of identity with the reference sequence. Thus, in one embodiment, homologous or derived sequences share at least 70 percent sequence identity. In a specific embodiment, homologous or derived sequences share at least 80 or 85 percent sequence identity. In a specific embodiment, homologous or derived sequences share at least 90 percent sequence identity. In a specific embodiment, homologous or derived sequences share at least 95 percent sequence identity. In a more specific embodiment, homologous or derived sequences share at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to a nucleic acid sequence under high-rigor hybridization conditions. Homologs with structural or functional similarity to a reference molecule can be chemical derivatives of the reference molecule. Methods for detecting, generating, and scanning structural and functional homologs as well as derivatives are known in the art.

[0117] Hybridization generally depends on the ability of denatured DNA to reanimate when complementary strands are present in an environment below its melting temperature. The greater the desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more difficult. Petition 870240080912, dated 09 / 23 / 2024, p. 50 / 136 44 / 105 more stringent, while lower temperatures are less stringent. For details and further explanations of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), incorporated herein by reference in its entirety.

[0118] The accuracy of hybridization reactions is easily determined by one skilled in the art and is generally an empirical calculation dependent on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.

[0119] Strict conditions or conditions of high rigor, as defined herein, may be identified by those which: (1) employ low ionic strength and high temperature for washing, for example, sodium chloride 0.015 M / sodium citrate 0.0015 M / sodium dedecyl sulfate at 0.1% 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) overnight hybridization in a solution employing 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, sonicated salmon sperm DNA (50 μL / ml), 0.1% SDS and 10% dextran sulfate at 42 °C, followed by a 10-minute wash at 42 °C in 0.2x SSC (sodium chloride / sodium citrate) followed by Petition 870240080912, dated 09 / 23 / 2024, page 51 / 136 45 / 105 minutes thorough washing consisting of 0.1 x SSC containing EDTA at 55 °C.

[0120] Each maximum numerical limitation given throughout this descriptive report is intended to include all lower numerical limitations, as if such lower numerical limitations were expressly written here. Each minimum numerical limitation given throughout this descriptive report will include all higher numerical limitations, as if such higher numerical limitations were expressly written here. Each numerical range given throughout this descriptive report will include all narrower numerical ranges that fall within such a broad numerical range, as if such narrower numerical ranges were all expressly written here.

[0121] As used herein, the term modification refers to an alteration that physically differentiates the modified molecule from the parent molecule. In one embodiment, an amino acid change in a variant polypeptide of CD47, HSVtk, EC-CD, or iCasp9 prepared according to the methods described herein differentiates it from the corresponding parent that has not been modified according to the methods described herein, such as wild-type proteins, a naturally occurring mutant protein, or another modified protein that does not include the modifications of such variant polypeptide. In another embodiment, a variant polypeptide includes one or more modifications that differentiate the function of the variant polypeptide from the unmodified polypeptide. For example, an amino acid change in a variant polypeptide affects its receptor binding profile. In other embodiments, a variant polypeptide comprises substitution, deletion, or other modifications. Petition 870240080912, dated 09 / 23 / 2024, page 52 / 136 46 / 105 insertion, or combinations thereof. In another embodiment, a variant polypeptide includes one or more modifications that increase its affinity for a receptor compared to the affinity of the unmodified polypeptide.

[0122] In one embodiment, a variant polypeptide includes one or more substitutions, insertions, or deletions relative to a corresponding native or parental sequence. In certain embodiments, a variant polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41 to 50 or 51 or more modifications.

[0123] By “episomal vector” is meant here a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; for example, it is not integrated into the genomic DNA of the host cell. Several episomal vectors are known in the art and are described below.

[0124] By knockout in the context of a gene means that the host cell harboring the knockout does not produce a functional protein product of the gene. As presented here, a knockout can result in a variety of ways, removing all or part of the coding sequence, introducing frameshift mutations so that a functional protein is not produced (truncated or nonsense sequence), removing or altering a regulatory component (e.g., a promoter) so that the gene is not transcribed, preventing translation through binding to mRNA, etc. Generally, the knockout is effected at the genomic DNA level, so that the descendants of the cells also carry the knockout permanently.

[0125] By knockin in the context of a gene Petition 870240080912, dated 09 / 23 / 2024, page 53 / 136 47 / 105 means that the host cell harboring the knockin has more active functional protein in the cell. As presented here, a knockin can be performed in several ways, usually by introducing at least one copy of a transgene (tg) encoding the protein into the cell, although this can also be achieved by replacing regulatory components as well, for example, by adding a constitutive promoter to the endogenous gene. In general, interference technologies result in the integration of the extra copy of the transgene into the host cell. VII. CELLS OF THE INVENTION

[0126] The invention provides compositions and methodologies for generating HIP cells, starting with wild-type cells, making them pluripotent (e.g., producing induced pluripotent stem cells, or iPSCs), and generating HIP cells from the iPSC population. A. METHODOLOGIES FOR GENETIC ALTERATIONS

[0127] The invention includes methods for modifying nucleic acid sequences within cells or under cell-free conditions to generate both pluripotent cells and HIP cells. Exemplary technologies include homologous recombination, knockin, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered interspace regularly clustered short palindromic repeats) / Cas9, and other site-specific nuclease technologies. These techniques allow for double-strand DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of Petition 870240080912, dated 09 / 23 / 2024, page 54 / 136 48 / 105 nucleic acid molecules, such as chromosomes, have endonucleases that recognize and bind to the sequences and induce a double-strand break in the nucleic acid molecule. The double-strand break is repaired by a non-homologous error-prone end joining (NHEJ) or by homologous recombination (HR).

[0128] As will be appreciated by those skilled in the art, several different techniques can be used to manipulate the pluripotent cells of the invention, as well as the manipulation of iPSCs to become hypoimmunogenic, as presented herein.

[0129] In general, these techniques can be used individually or in combination. For example, in the generation of HIP cells, CRISPR can be used to reduce the expression of the B2M and / or active CIITA protein in the manipulated cells, with viral techniques (e.g., lentivirus) to perform knockin of CD47 functionality. Also, as will be appreciated by those skilled in the art, although one modality sequentially uses a CRISPR step to perform B2M knockout, followed by a CRISPR step to perform CIITA knockin with a final lentivirus step to perform knockin on CD47 functionality, these genes can be manipulated in different orders using different technologies.

[0130] As discussed in more detail below, transient expression of reprogramming genes is generally performed to generate / induce pluripotent stem cells. A. CRISPR TECHNOLOGIES

[0131] In one form, the cells are Petition 870240080912, dated 09 / 23 / 2024, page 55 / 136 49 / 105 manipulated using clustered regularly spaced short palindromic repeats (CRISPR) / Cas technology, as it is known in the art. CRISPR can be used to generate the starter iPSCs or to generate HIP cells from the iPSCs. There are a large number of CRISPR-based techniques; see, for example, Doudna and Charpentier, Science doi:10.1126 / science.1258096, incorporated here by reference. CRISPR techniques and kits are commercially available. B. TALEN TECHNOLOGIES

[0132] In some embodiments, the HIP cells of the invention are produced using Transcription Activator Effector Nuclease (TALEN) methodologies. TALEN are restriction enzymes combined with a nuclease that can be modified to bind to and cut virtually any desired DNA sequence. TALEN kits are commercially available. C. ZINC FINGER TECHNOLOGIES

[0133] In one embodiment, cells are manipulated using Zn finger nuclease technologies. Zn finger nucleases are artificial restriction enzymes generated by fusing a DNA-binding domain of the zinc finger to a DNA-cleavage domain. Zinc finger domains can be manipulated to target specific desired DNA sequences, and this allows zinc finger nucleases to target single sequences within complex genomes. By leveraging endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALENs. Petition 870240080912, dated 09 / 23 / 2024, page 56 / 136 50 / 105 D. VIRUS-BASED TECHNOLOGIES

[0134] There is a wide variety of viral techniques that can be used to generate the HIP cells of the invention (as well as for the original generation of iPSCs), including, without limitation, the use of retroviral vectors, lentiviral vectors, adenovirus vectors, and Sendai viral vectors. The episomal vectors used in the generation of iPSCs are described below. E. Down-regulation of genes using RNA interference

[0135] In other modalities, genes encoding proteins used in HLA molecules are negatively regulated by RNAi technologies. RNA interference (RNAi) is a process in which RNA molecules inhibit gene expression, usually by causing the degradation of specific mRNA molecules. Two types of RNA molecules—microRNA (miRNA) and small interfering RNA (siRNA)—are central to RNA interference. They bind to target mRNA molecules and increase or decrease their activity. RNAi helps cells defend themselves against parasitic nucleic acids, such as those from viruses and transposons. RNAi also influences development.

[0136] sdRNA molecules are a class of asymmetric siRNAs comprising a guide (antisense) strand of 19-21 bases. They contain pyrimidines modified with 5'-phosphate, 2'Ome or 2'F and six phosphothioates in the 3' positions. They also contain a sensitive chain containing 3' sterol-conjugated moieties, two phosphorioates at the 3' position, and pyrimidines modified with 2'Ome. Both chains contain 2'Ome purines with continuous stretches of unmodified purines that do not Petition 870240080912, dated 09 / 23 / 2024, page 57 / 136 51 / 105 exceed a length of 3. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety.

[0137] For all these technologies, well-known recombinant techniques are used to generate recombinant nucleic acids, as presented here. In certain embodiments, recombinant nucleic acids (which may encode a desired polypeptide, for example, CD47, or break sequences) can be operatively linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell and subject to treatment. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.Typically, one or more regulatory nucleotide sequences may include, without limitation, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters, as known in the art, are also contemplated. Promoters may be naturally occurring promoters or hybrid promoters that combine elements from more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or an expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain embodiments include an expression vector comprising a... Petition 870240080912, dated 09 / 23 / 2024, page 58 / 136 52 / 105 nucleotide sequence encoding a variant polypeptide operatively linked to at least one regulatory sequence. The regulatory sequence for use here includes promoters, enhancers, and other expression control elements. In certain embodiments, an expression vector is designed for choice of the host cell to be transformed, the specific variant polypeptide to be expressed, the number of copies of the vector, the ability to control that number of copies, or the expression of any other protein encoded by the vector, such as antibiotic markers.

[0138] Examples of suitable mammalian promoters include, for example, promoters of the following genes: hamster ubiquitin / S27a promoter (WO 97 / 15664), simian vaccine vacuolation virus early promoter (SV40), adenovirus late major promoter, mouse metallothione-I promoter, Rous Sarcoma Virus (RSV) long terminal repeat region, mouse mammary tumor virus (MMTV) promoter, Moloney murine leukemia virus long terminal repeat region, and human cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters are actin, immunoglobulin, or heat shock promoter(s).

[0139] In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyomavirus, chicken poxvirus (UK 2211504 published July 5, 1989), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and Simian Virus 40 (SV40). In other embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, Petition 870240080912, dated 09 / 23 / 2024, page 59 / 136 53 / 105 an immunoglobulin promoter and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also contains the viral replication origin of SV40. Fiers et al., Nature 273: 113-120 (1978). The immediate initial promoter of human cytomegalovirus is conveniently obtained as a HindIIIE restriction fragment. Greenaway, PJ et al., Gene 18: 355-360 (1982). The preceding references are incorporated by reference in their entirety. B. Generation of Pluripotent Cells

[0140] The invention provides methods for producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to provide the pluripotent stem cells.

[0141] The generation of mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those skilled in the art, there are a variety of different methods for the generation of iPSCs. The original induction was performed from embryonic or adult mouse fibroblasts using viral introduction of four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4; see Takahashi and Yamanaka Cell 126: 663-676 (2006), incorporated herein by reference in its entirety and specifically for the techniques outlined therein. Since then, several methods have been developed; See Seki et al., World J. Stem Cells 7 (1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, which are here. Petition 870240080912, dated 09 / 23 / 2024, page 60 / 136 54 / 105 expressly incorporated by reference in their entirety, and in particular for the methods of generating hiPSCs (see, for example, Chapter 3 of that last reference).

[0142] Generally, iPSCs are generated by the transient expression of one or more “reprogramming factors” in the host cell, usually introduced using episomal vectors. Under these conditions, small quantities of cells are induced to become iPSCs (in general, the efficiency of this step is low, since no selection marker is used). Once the cells are reprogrammed and become pluripotent, they lose the episomal vector and produce the factors using endogenous genes. This loss of the episomal vector (or vectors) results in cells called “zero footprint” cells. This is desirable because the fewer genetic modifications (particularly in the host cell genome), the better. Thus, it is preferable that the resulting hiPSCs have no permanent genetic modifications.

[0143] As is also appreciated by those skilled in the art, the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of transforming cells into a pluripotent state decreases, just as “pluripotency,” for example, fewer reprogramming factors may result in cells that are not fully pluripotent, but may only have the ability to differentiate into fewer cell types.

[0144] In some modalities, a single reprogramming factor, OCT4, is used. In other modalities, two reprogramming factors, OCT4 and KLF4, are used. In other modalities, three factors are used. Petition 870240080912, dated 09 / 23 / 2024, page 61 / 136 55 / 105 reprogramming, OCT4, KLF4 and SOX2. In other modalities, four reprogramming factors are used: OCT4, KLF4, SOX2 and c-Myc. In other modalities, 5, 6 or 7 reprogramming factors selected from SOKMNLT antigens may be used; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28 and SV40L T antigens.

[0145] In general, these reprogramming factor genes are provided in episomal vectors as known in the art and are commercially available. For example, ThermoFisher / Invitrogen sells a sendai virus reprogramming kit for zero-footprint hiPSC generation, see catalog number A34546. ThermoFisher also sells EBNA-based systems, see catalog number A14703.

[0146] In addition, there are a number of commercially available hiPSC lines; see, for example, the Gibco® Episomal HiPSC line, K18945, which is a zero-footprint, virus-free human iPSC cell line (see also Burridge et al., 2011, supra).

[0147] In general, as is known in the art, iPSCs are produced from non-pluripotent cells, such as CD34+ cord blood cells, fibroblasts, etc., transiently expressing the reprogramming factors as described herein.

[0148] For example, successful iPSCs were also generated using only Oct3 / 4, Sox2 and Klf4, while omitting C-Myc, although with reduced reprogramming efficiency.

[0149] In general, iPSCs are characterized by the expression of certain factors including KLF4, Nanog, OCT4, Petition 870240080912, dated 09 / 23 / 2024, page 62 / 136 56 / 105 SOX2, ESRRB, TBX3, c-Myc, and TCL1. The novel or enhanced expression of these factors for the purposes of the invention may be via induction or modulation of an endogenous locus or from expression from a transgene.

[0150] For example, murine iPSCs can be generated using the methods of Diecke et al., Sci Rep. 2015, January 28; 5: 8081 (doi:10.1038 / srep08081), incorporated herein by reference in its entirety and specifically for the methods and reagents for the generation of miPSCs. See also, for example, Burridge et al., PLoS One, 2011 6(4): 18293, incorporated herein by reference in its entirety and specifically for the methods presented herein.

[0151] In some cases, cell pluripotency is measured or confirmed as presented here, for example, through the assay of reprogramming factors, as generally shown in Figure 17, or through the performance of differentiation reactions, as outlined here and in the Examples. C. GENERATION OF HYPOIMMUNOGENIC PLURIPOTENT CELLS

[0152] The present invention is directed to the generation, manipulation, growth and transplantation of hypoimmunogenic cells in a patient as defined herein. The generation of HIP cells from pluripotent cells is performed with only three genetic alterations, resulting in minimal disruption of cellular activity, but conferring immunosilencing to the cells.

[0153] As discussed here, one approach utilizes a reduction or elimination of MHC I and II protein activity (HLA I and II when the cells are human). This can be accomplished by altering the genes that encode their Petition 870240080912, dated 09 / 23 / 2024, page 63 / 136 57 / 105 components. In one modality, the coding region or regulatory sequences of the gene are disrupted using CRISPR. In another modality, gene translation is reduced using RNA interference technologies. The third change is a change in a gene that regulates macrophage susceptibility to phagocytosis, such as CD47, and this is usually a knockin of a gene using viral technologies.

[0154] In some cases, where CRISPR is being used for genetic modifications, hiPSC cells containing a Cas9 construct that allows for high-efficiency cell line editing can be used; see, for example, the Human Episomal Cas9 iPSC cell line, A33124, from Life Technologies. 1. HLA-I Reduction

[0155] The HIP cells of the invention include a reduction in MHC I function (HLA I when the cells are derived from human cells).

[0156] As will be appreciated by experts in the art, reduction of function can be achieved in several ways, including removing nucleic acid sequences from a gene, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with nonsense sequences, structure-change mutations can be made, all or part of a regulatory sequence, such as a promoter, can be removed or replaced, initiation sequences can be removed or replaced, etc.

[0157] As will be understood by those skilled in the art, the successful reduction of MHC I function (HLA I Petition 870240080912, dated 09 / 23 / 2024, p. 64 / 136 58 / 105 when the cells are derived from human cells) in pluripotent cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-A, B, C antibodies that bind to the alpha chain of human major histocompatibility class I HLA antigens. a. B2M CHANGE

[0158] In one embodiment, the reduction of HLA-I activity is achieved by disrupting the expression of the microglobulin 2-2 gene in the pluripotent stem cell, whose human sequence is disclosed herein. This alteration is generally referred to here as a gene knockout, and in the HIP cells of the invention it is performed on both alleles in the host cell. Generally, the techniques for making the two disruptions are the same.

[0159] One particularly useful modality uses CRISPR technology to disrupt the gene. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of the gene, so that no functional protein is produced, often the result of frameshift mutations that result in the generation of stop codons so that truncated, non-functional proteins are produced.

[0160] Consequently, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mice or the B2M gene in humans. After gene editing, the transfected iPSC cultures are dissociated into individual cells. The Petition 870240080912, dated 09 / 23 / 2024, page 65 / 136 59 / 105 individual cells are expanded into full-size colonies and tested for CRISPR editing by scanning for the presence of aberrant sequences from the CRISPR cleavage site. Clones with deletions in both alleles are scouted. Such clones did not express B2M / B2M as demonstrated by PCR and did not express HLA-I as demonstrated by FACS analysis (see examples 1 and 6, for example).

[0161] Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies to the B2M protein. In another embodiment, reverse transcriptase polymerase chain reactions (rt-PCR) confirm the presence of the inactivating alteration.

[0162] In addition, cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This can be assayed by FACS analysis using antibodies to one or more HLA cell surface components, as discussed above.

[0163] It is worth noting that others have had poor results when attempting to silence the B2M genes in both alleles. See, for example, Gornalusse et al., Nature Biotech. Doi / 10.1038 / nbt.3860).

[0164] In addition to a reduction in HLA I, the cells The HIPs of the invention also lack MHC II function (HLA II when the cells are derived from human cells).

[0165] As will be appreciated by those skilled in the art, reduction of function can be achieved in several ways, including removing nucleic acid sequences from a gene, adding nucleic acid sequences to a gene, breaking the reading frame, interrupting the sequence. Petition 870240080912, dated 09 / 23 / 2024, p. 66 / 136 60 / 105 with other sequences, or by altering the regulatory components of the nucleic acid. In one embodiment, all or part of a coding region of the gene of interest may be removed or replaced by “nonsense” sequences. In another embodiment, regulatory sequences, such as a promoter, may be removed or replaced, translation initiation sequences may be removed or replaced, etc.

[0166] The successful reduction of MHC II function (HLA II when cells are derived from human cells) in pluripotent cells or their derivatives can be measured using techniques known in the art such as Western blotting using antibodies to the protein, FACS techniques, rt-PCR, etc. a. CIITA CHANGE

[0167] In one embodiment, the reduction of HLA-II activity is achieved by disrupting the expression of the CIITA gene in the pluripotent stem cell, the human sequence of which is illustrated herein. This alteration is generally referred to herein as a gene “knockout,” and in the HIP cells of the invention it is performed on both alleles in the host cell.

[0168] Assays to test whether the CIITA gene has been inactivated are known and described here. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies to the CIITA protein. In another embodiment, reverse transcriptase polymerase chain reactions (rtPCR) confirm the presence of the inactivating alteration.

[0169] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is performed as is known in the art (see Figure 21, for example) and Petition 870240080912, dated 09 / 23 / 2024, page 67 / 136 61 / 105 is usually performed using Western Blots or FACS analysis based on commercial antibodies that bind to human HLA-II HLA-DR, DP antigens, and most DQ antigens as described below.

[0170] A particularly useful approach utilizes CRISPR technology to disrupt the CIITA gene. CRISPRs were designed to target the coding sequence of the Ciita gene in mice or the CIITA gene in humans, a transcription factor essential for all MHC II molecules. Following gene editing, transfected iPSC cultures were dissociated into single cells. These were expanded to full-size colonies and tested for successful CRISPR editing by screening for the presence of an aberrant sequence from the CRISPR cleavage site. Clones with deletions do not express Ciita / CIITA as determined by PCR and do not express MHC II / HLA-II as determined by FACS analysis. 3. REDUCTION OF PHAGOCYTOSIS

[0171] In addition to HLA I and II (or MHC I and II) reduction, generally with the use of B2M and CIITA knockouts, the HIP cells of the invention have a reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting HIP cells escape the immune macrophage and innate pathways due to one or more CD47 transgenics. A. INCREASE IN CD47

[0172] In some embodiments, reduced macrophage phagocytosis and NK cell susceptibility to death result from increased CD47 on the HIP cell surface. This is accomplished in various ways, as will be appreciated by those versed in the technique using knockin or transgenic technologies. In some cases, increased CD47 expression Petition 870240080912, dated 09 / 23 / 2024, p. 68 / 136 62 / 105 results from one or more CD47 transgenes.

[0173] Consequently, in some forms, one or more copies of a CD47 gene are added to the cells. HIP under the control of an inducible or constitutive promoter, the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. CD47 genes can integrate into the host cell genome under the control of a suitable promoter as known in the art.

[0174] HIP cell lines were generated from B2M- / - CIITA- / - iPSCs. Cells containing lentivirus vectors expressing CD47 were selected using a blasticidin marker. The CD47 gene sequence was synthesized and the DNA was cloned into the pLentivirus pLenti6 / V5 plasmid with blasticidin resistance (Thermo Fisher Scientific, Waltham, MA, USA).

[0175] In some modalities, CD47 gene expression can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by replacing the endogenous promoter with a constitutive promoter or a different inducible promoter. This can usually be accomplished using known techniques such as CRISPR.

[0176] Once altered, the presence of sufficient CD47 expression can be assayed using known techniques such as those described in the Examples, such as Western blots, ELISA assays, or FACS assays using anti-CD47 antibodies. In general, sufficiency in this context means an increase in CD47 expression on the HIP cell surface that silences NK cell death. Natural expression levels in cells are too low to protect Petition 870240080912, dated 09 / 23 / 2024, page 69 / 136 63 / 105 the same against NK cell lysis, once their MHC I is removed. 4. Suicide Genes

[0177] In some embodiments, the invention provides hypoimmunogenic pluripotent cells comprising a “suicide gene” or “suicide switch.” These are incorporated to function as a “safety switch” that can cause the death of hypoimmunogenic pluripotent cells if they grow and divide in an undesirable manner. The “suicide gene” ablation approach involves a suicide gene in a gene transfer vector that encodes a protein that results in cell death only when activated by a specific compound. A suicide gene may encode an enzyme that selectively converts a non-toxic compound into highly toxic metabolites. The result is specifically to eliminate the cells that express the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir.In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase gene (EC-CD) and the stimulus is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap 20 (10): 1932-1943 (2012), Xu et al., Cell Res. 8: 73-8 (1998), both incorporated herein by reference in their entirety).

[0178] In other embodiments, the suicide gene is an inducible Caspase protein. An inducible caspase protein comprises at least one portion of a Caspase protein with the ability to induce apoptosis. In one embodiment, the portion of the Caspase protein is exemplified in SEQ ID NO: 6. In preferred embodiments, the inducible Caspase protein is iCasp9. It comprises the sequence of the binding protein. Petition 870240080912, dated 09 / 23 / 2024, p. 70 / 136 64 / 105 a human FK50 6, FKBP12, with an F36V mutation, linked through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small molecule dimerizing agent, AP1903. Thus, the suicide function of iCasp9 in the present invention is triggered by the administration of a chemical dimerization inducer (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization causes rapid induction of apoptosis. (See document WO2011146862; Stasi et al, N. Engl. J. Med 365; 18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13: 913-924 (2007), each is incorporated herein by reference in its entirety). 5. Assays for HIP Phenotypes and Pluripotentiality Retention

[0179] Once HIP cells have been generated, they can be analyzed for their hypoimmunogenicity and / or retention of pluripotency, as is generally described here and in the examples.

[0180] For example, hypoimmunogenicity is assayed using various techniques as exemplified in Figure 13 and Figure 15. These techniques include transplantation into allogeneic hosts and monitoring the growth of HIP cells (e.g., teratomas) that escape the host immune system. HIP derivatives are transduced to express luciferase and can be followed using bioluminescence imaging. Similarly, the response of host animal T cells and / or B cells to HIP cells is tested to confirm that HIP cells do not cause an immune reaction in the host animal. T cell function is assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). A Petition 870240080912, dated 09 / 23 / 2024, page 71 / 136 65 / 105 B cell response or antibody response is assessed using FACS or Luminex. Additionally or alternatively, cells can be tested for their ability to evade innate immune responses, for example, NK cell death, as is generally shown in Figure 14. The lipolytic activity of NK cells is assessed in vitro or in vivo (as shown in Figure 15).

[0181] Similarly, the retention of pluripotency is tested in several ways. In one embodiment, pluripotency is assayed by the expression of certain specific pluripotency factors, as generally described herein and shown in Figure 29. Additionally or alternatively, HIP cells are differentiated into one or more cell types as an indication of pluripotency. d. PREFERRED EMBODIMENTS OF THE INVENTION

[0182] Hypoimmunogenic pluripotent stem cells (“HIP cells”) are provided here that exhibit pluripotency but do not result in a host immune response when transplanted into an allogeneic host, such as a human patient, either as HIP cells or as differentiated products of HIP cells.

[0183] In one embodiment, human pluripotent stem cells (hiPSCs) are made hypoimmunogenic by a) disruption of the B2M gene in each allele (e.g., B2M / -), b) disruption of the CIITA gene in each allele (e.g., CIITA- / -), and c) overexpression of the CD47 gene (CD47+, for example, by introducing one or more additional copies of the CD47 gene or activating the genomic gene). This renders the hiPSC population B2M- / - CIITA- / - CD47tg. In a preferred embodiment, the cells are not immunogenic. In another Petition 870240080912, dated 09 / 23 / 2024, page 72 / 136 In the 66 / 105 modality, the HIP cells are rendered non-immunogenic as described above, but are further modified by the inclusion of an induced suicide gene that is induced to kill the cells in vivo when necessary. e. MAINTENANCE OF HIP CELLS

[0184] Once generated, HIP cells can be maintained in an undifferentiated state, as is known by iPSC maintenance. For example, HIP cells are cultured in Matrigel using a culture medium that prevents differentiation and maintains pluripotency. f. Differentiation of hippocampus cells

[0185] The invention provides HIP cells that are differentiated into different cell types for subsequent transplantation into individuals. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques. The cells are differentiated in suspension and then placed in a gel matrix form, such as Matrigel, gelatin, or fibrin / thrombin forms to facilitate cell survival. Differentiation is assayed as is known in the art, generally by assessing the presence of specific cell markers.

[0186] In some modalities, HIP cells are differentiated into hepatocytes to address hepatocyte dysfunction or liver cirrhosis. There are several techniques that can be used to differentiate cells. HIP in hepatocytes; see for example Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods Mol Biol 698: 305-314 (2011), Si-Tayeb et al., Hepatology 51: 297-305 (2010) and Asgari et al., Stem Cell Rev (: 493-504 (2013), all Petition 870240080912, dated 09 / 23 / 2024, page 73 / 136 67 / 105 which are hereby expressly incorporated by reference in their entirety and specifically for the methodologies and reagents for differentiation. Differentiation is assayed as is known in the art, generally by assessing the presence of hepatocyte-associated and / or specific markers, including, without limitation, albumin, alpha-fetoprotein and fibrinogen. Differentiation may also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage.

[0187] In some embodiments, HIP cells are differentiated into beta-like cells or islet organoids for transplantation to treat type I diabetes mellitus (T1DM). Cellular systems are a promising way to address T1DM, see, e.g., Ellis et al., Doi / 10.1038 / nrgastro.2017.93, incorporated here by reference. In addition, Pagliuca et al. reports the successful differentiation of β cells from hiPSCs (see doi / 10.106 / j.cell.2014.09.040, incorporated here by reference in its entirety and in particular for the methods and reagents presented here for the large-scale production of human β cells from human pluripotent stem cells). Furthermore, Vegas et al. shows the production of human β cells from human pluripotent stem cells, followed by encapsulation to avoid immune rejection by the host; (doi:10.1038 / nm.4030, incorporated herein by reference in its entirety and in particular for the methods and reagents set forth therein for the large-scale production of functional human stem cells (human pluripotent stem cells).

[0188] Differentiation is rehearsed as it is Petition 870240080912, dated 09 / 23 / 2024, page 74 / 136 68 / 105 known in the art, generally assessing the presence of associated or specific β-cell markers, including, without limitation, insulin. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, doi:10.1016 / j.cell.2016.09.002, incorporated herein by reference in its entirety, and specifically for the biomarkers presented herein.

[0189] Once dHIP beta cells are generated, they can be transplanted (as a cell suspension or within a gel matrix as discussed here) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, a muscle, or subcutaneous pouches.

[0190] In some modalities, HIP cells are differentiated into retinal pigment epithelium (RPE) to address vision-threatening diseases of the eye. Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al., Stem Cell Reports 2014: 2: 205-18, incorporated herein by reference in its entirety and in particular for the methods and reagents presented therein for the differentiation techniques and reagents; see also Mandai et al., doi:10.1056 / NEJMoa1608368, also incorporated in its entirety for RPE cell slide generation and transplantation techniques in patients.

[0191] Differentiation can be assayed as is known in the art, generally by assessing the presence of associated and / or specific PSE markers or by measuring functionally. See, for example, Kamao et al., Doi:10.1016 / j.stemcr.2013.12.007, incorporated herein by reference in its entirety and specifically for the Petition 870240080912, dated 09 / 23 / 2024, page 75 / 136 69 / 105 markers presented in the first paragraph of the results section.

[0192] In some modalities, HIP cells are differentiated into cardiomyocytes to treat cardiovascular diseases. Techniques are known in the art for the differentiation of hiPSCs into cardiomyocytes and are discussed in the Examples. Differentiation can be assayed as is known in the art, generally by assessing the presence of cardiomyocyte-associated or specific markers or by measuring functionally; see for example Loh et al., doi:10.1016 / j.cell.2016.06.001, incorporated herein by reference in its entirety and specifically for stem cell differentiation methods including cardiomyocytes.

[0193] In some embodiments, HIP cells are differentiated into endothelial colony-forming cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., doi:10.1038 / nbt.3048, incorporated by reference in its entirety and specifically for methods and reagents for the generation of endothelial cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as is known in the art, generally by assessing the presence of endothelial cell-associated or specific markers or by measuring functionally.

[0194] In some modalities, HIP cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones to address autoimmune thyroiditis. Techniques for differentiating Petition 870240080912, dated 09 / 23 / 2024, page 76 / 136 70 / 105 thyroid cells are known in the art. See, for example, Kurmann et al., doi:10.106 / j.stem.2015.09.004, herein incorporated by reference in its entirety and specifically for methods and reagents for generating thyroid cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as is known in the art, generally by assessing the presence of thyroid cell-associated or specific markers or by measuring functionally. g. Transplantation of differentiated HIP cells

[0195] As will be appreciated by those skilled in the art, the differentiated HIP derivatives are transplanted using techniques known in the art that depend both on the cell type and the end use of these cells. In general, the dHIP cells of the invention are transplanted intravenously or by injection into particular locations in the patient. When transplanted into specific locations, the cells can be suspended in a gel matrix to prevent dispersion while they attach.

[0196] In order that the invention described herein may be more fully understood, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting this invention in any way. viii. EXAMPLES a. GENERAL TECHNIQUES 1. Generation of IPSCs from MICE

[0197] These cells were generated using the methods of Diecke et al, Sci Rep. 2015, January 28; 5:8081 (doi:10.1038 / srep08081), incorporated herein in its entirety and Petition 870240080912, dated 09 / 23 / 2024, page 77 / 136 71 / 105 specifically for methods and reagents for the generation of miPSCs.

[0198] Mouse tail fibroblasts were dissociated and isolated with type IV collagenase (Life Technologies, Grand Island, NY, USA) and maintained with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), L-glutamine, 4.5 g / l glucose, 100 U / ml penicillin and 100 μg / ml streptomycin at 37 °C, O2 20% and 5% CO2 in a humidified incubator. One χ¹⁰⁶ murine fibroblast was then reprogrammed using a novel codon-optimized mini-intronic plasmid (co-MIP) (10–12 pm of DNA) expressing the four reprogramming factors Oct4, KLF4, Sox2, and c-Myc using the Neon Transfection system. After transfection, fibroblasts were plated onto a MEF feeder layer and maintained in fibroblast media with the addition of sodium butyrate (0.2 mM) and 50 μg ascorbic acid / ml. When colonies appeared as CES-, the medium was changed to murine iPSC medium containing DMEM, 20% FBS, L-glutamine, non-essential amino acids (NEAA), mercaptoethanol β, and 10 ng / ml leukemia inhibitory factor (LIF). After 2 passages, murine iPSCs were transferred to 0.2% gelatin-coated plates and subsequently expanded. With each passage, iPSCs were graded for the murine pluripotency marker SSEA-1 using magnetically activated cell sorting (MACS). 2. GENERATION OF HUMAN IPSCs

[0199] The generation of hiPSCs was performed as generally described in Burridge et al., PLoS One, 2011 6(4):18293, incorporated herein by reference in its entirety and specifically for the methods described herein. Petition 870240080912, dated 09 / 23 / 2024, page 78 / 136 72 / 105

[0200] The Gibco® Human Episomal iPSC Line (Catalog No. A33124, Thermo Fisher Scientific) was derived from CD34+ cord blood using a three-plasmid, seven-factor EBNA-based episomal system (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen). This iPSC line is considered zero footprint, as there was no genome integration from the reprogramming event. It has been shown to be free of all reprogramming genes.

[0201] The Gibco® Human Episomal iPSC Line has a normal karyotype and endogenous expression of pluripotent markers such as Oct4, Sox2, and Nanog (as shown by RT-PCR) and Oct4, SSEA4, TRA-1-60, and TRA-1-81 (as shown by ICC). Whole genome expression and epigenetic profile analyses have demonstrated that this episomal hiPSC line is molecularly indistinguishable from human embryonic stem cell lines (Burridge et al., 2011). In directed differentiation and teratoma analyses, these hiPSCs maintained their differentiation potential for ectodermal, endodermal, and mesodermal lineages (Burridge et al., 2011). Furthermore, vascular, hematopoietic, neural, and cardiac lineages were derived with robust efficiencies (Burridge et al., 2011). 3. FACS ANALYSIS OF SURFACE MOLECULES a. DETECTION OF HUMAN HLA I SURFACE MOLECULES

[0202] Human iPSCs, iCMs and iECs were plated in 6-well plates and stimulated with 100 ng / ml of human IFNγ (Peprotech, Rocket Hill, NJ). Cells were collected and labeled with HLA-A, B, C antibody conjugated with APC (clone G46_2.6, category no. 562006, BD BioSciences, San Jose, CA, USA) or IgG1 isotype control antibody Petition 870240080912, dated 09 / 23 / 2024, page 79 / 136 73 / 105 conjugated with APC (clone MOPC-21, category no. 555751, BD (BioSciences). The HLA-A, B, C antibody binds to the alpha chain of human major histocompatibility class I HLA antigens. Data analysis was performed by flow cytometry (BD Bioscience) and results were expressed as fold change for the isotype control. 4. DETECTION OF HUMAN HLA II SURFACE MOLECULES

[0203] Human iPSCs, iCMs, and iECs were plated in 6-well plates and stimulated with 100 ng / ml of human TNFα (Peprotech, Rocket Hill, NJ). Cells were collected and labeled with Alexa-fluor647-labeled HLA-DR, DP, DQ antibody (clone Tu3a, category no. 563591, BD BioSciences, San Jose, CA, USA) or Alexa-fluor647-labeled IgG2a isotype control antibody (clone G155-178, category no. 557715, BD BioSciences). The HLA-DR, DP, DQ antibody binds to human HLA-DR, HLA-DR, DP antigens and most DQ antigens. Data analysis was performed by flow cytometry (BD Bioscience) and results were expressed as fold change for the isotype control. 5. DETECTION OF HUMAN CD47 SURFACE MOLECULES

[0204] Human IPSCs, iCMs and iECs were plated in 6-well plates and stimulated with 100 ng / ml of Human IFNγ (Peprotech, Rocket Hill, NJ). Cells were collected and labeled with CD47 conjugated with PerCP-Cy5 (clone B6H12, category number 561261, BD BioSciences, San Jose, CA, USA) or IgG1 isotype control antibody conjugated with PerCP-Cy5 (clone MOPC-21, category number 550795, BD BioSciences). The B6H12 CD47 monoclonal antibody binds specifically to CD47, an N-linked glycan protein of Petition 870240080912, dated 09 / 23 / 2024, page 80 / 136 74 / 105 42-52 kDa. Data analysis was performed by flow cytometry (BD Bioscience) and results were expressed as fold change for the isotype control. 6. DETECTION OF MHC I SURFACE MOLECULES FROM MICE

[0205] For the detection of MHC I surface molecules in miPSC, miEC, miSMC, and miCM, cells were plated in 6-well gelatin-coated plates and stimulated with 100 ng / ml of mouse IFNγ (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with PerCP-eFlour710-labeled MHCI antibody (clone AF688.5.5.3, cat. 46-5958-82, eBioscience, Santa Clara, CA, USA) or PerCP-eFlour710-labeled IgG2b isotype control antibody (clone eB149 / 10H5, cat. 46-4031-80, eBioscience). The MHCI antibody reacts with the MHC class I H-2Kb alloantigen. Data analysis was performed by flow cytometry (BD Bioscience) and results were expressed as fold change for the isotype control. 7. DETECTION OF MHC II SURFACE MOLECULES MOUSE

[0206] For the detection of MHC II surface molecules in miPSC, miEC, miSMC, and miCM, cells were plated in 6-well gelatin-coated plates and stimulated with 100 ng / ml of mouse TNFα (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with PerCP-eFlour710-labeled MHC II antibody (clone M5 / 114.15.2, cat. 46-5321-82, eBioscience, Santa Clara, CA) or PerCP-eFlour710-labeled IgG2α / K isotype control antibody (clone eBM2a, cat. 46-4724-80, eBioscience). The MHC II antibody reacts with the complex of Petition 870240080912, dated 09 / 23 / 2024, page 81 / 136 75 / 105 mouse major histocompatibility class II, both glycoproteins encoded in subregions IA and IE. Data analysis was performed by flow cytometry (BD). Bioscience) and the results were expressed as fold change for the isotype control. 8. DETECTION OF CD47 SURFACE MOLECULES FROM MICE

[0207] For the detection of surface molecules In miPSC, miEC, miSMC, and miCM cells, Cd47 cells were plated in 6-well gelatin-coated plates and stimulated with 100 ng / ml mouse IFNγ (Peprotech, Rocket Hill, NJ, USA). After collection, cells were labeled with Alexa Fluor 647-labeled Cd47 antibody (clone miap301, category no. 563584, BD BioSciences, San Jose, CA, USA) or Alexa Fluor 647-labeled IgG2a / K isotype control antibody (clone R35-95, category no. 557690, BD BioSciences). The Cd47 antibody binds specifically to the extracellular domain of CD47, also known as Integrin-Associated Protein (IAP). Data analysis was performed by flow cytometry (BD Bioscience) and results were expressed as fold change for the isotype control. 9. Determining Cellular Morphology of Mice in Vivo After Allogeneic Transplantation

[0208] Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 mi cells, miPSC-derived cardiomyocytes (miCM), miPSC-derived smooth muscle cells (miSMC), or miPSC-derived endothelial cells (miEC) in 250 µl of 0.9% saline solution were mixed with 250 µl of BD Matrigel High Concentration (1:1 BD Biosciences) Petition 870240080912, dated 09 / 23 / 2024, p. 82 / 136 76 / 105 and injected subcutaneously in the lower dorsal region of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding. A 5 gm thick section was cut and stained with Hematoxylin and Eosin (HE). 10. DETERMINATION OF HUMAN CELL MORPHOLOGY IN VIVO AFTER ALLOGENEIC TRANSPLANTATION

[0209] Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million cells, hiPSC-derived cardiomyocytes (hiCM) or hiPSC-derived endothelial cells (hiEC) in 250 µL of 0.9% saline solution containing ZVAD (100 mM, benzyloxycarbonyl-ValAla-Asp(O-methyl)-fluoromethylketone, Calbiochem), Bcl-XL BH4 (cell-permeated TAT peptide, 50 nM, Calbiochem), cyclosporine A (200 nM, Sigma), IGF-1 (100 ng / ml, Peprotech), and pinacidil (50 mM, Sigma) were mixed with 250 µL of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 2, 4, Samples were placed 6, 8, 10, and 12 weeks after implantation and fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding. A 5 g thick section was cut and stained with Hematoxylin and Eosin (HE). b. EXAMPLE 1: GENERATION OF PLURIPOTENT β-2 MICROGLOBULIN KNOCKOUT CELLS IN A MOUSE MODEL

[0210] Induced Pluripotent Cell Generation: Petition 870240080912, dated 09 / 23 / 2024, page 83 / 136 77 / 105 hypoimmune pluripotent cells were generated in a mouse modality. Human hypoimmune pluripotent cells are another modality that are generated using the strategies described here.

[0211] Mouse-induced pluripotent stem cells (miPSCs) were generated from C57BL / 6 fibroblasts. Mitomycin-inhibited CF1 mouse embryonic fibroblasts (MEF, Applied Stemcell, CA) were thawed and maintained in DMEM + GlutaMax 31966 (Gibco, Grand Island, NY, USA) with 10% heat-inactivated fetal calf serum (FCS hi), 1% MEM-NEAA, and 1% Pen Strep (Thermo Fisher Scientific, Gibco, Waltham, MA, USA). After MEF feeder cells formed a 100% confluent monolayer, miPSCs were cultured in MEF in KO DMEM 10829 with serum replacement of 15% KO, 1% MEM-NEAA, 1% Pen Strep (Thermo Fisher-Gibco), 1x beta-mercaptoethanol, and 100 LIF units (Millipore, Billerica, MA, USA). Cells were maintained in 10 cm plates, the medium was changed daily, and cells were passed every 2-3 days using 0.05% Trypsin-EDTA (Thermo Fisher-Gibco).The miPSCs were cultured in gelatin (Millipore) without feeders using conventional media. The cell cultures were regularly analyzed for mycoplasma infections using the MycoAlert Kit (Lonza, Cologne, Germany).

[0212] Mice: BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU, CAnN. CgFoxn1 / / Crl, H2d) and light beige (CBySmn. CB17-Prkdcscid / J) (all 6-12 weeks old) were used as recipients for different assays (all 6-12 weeks old). The mice were purchased from Charles River Laboratories. Petition 870240080912, dated 09 / 23 / 2024, page 84 / 136 78 / 105 (Sulzfeld, Germany) and received humane care in accordance with the Guidelines for the Principles of Laboratory Animals. The animal experiments were approved by the Hamburg Office for Health and Consumer Protection and carried out in accordance with local and EU guidelines.

[0213] Confirmation of Pluripotency: Pluripotency was demonstrated by rtPCR. RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific). A DNase I step was included to remove the Contaminating genomic DNA. cDNA was generated using the Applied Biosystems® High-Throughput cDNA Reverse Transcription Kit. Non-reverse transcriptase (no RT) controls were also generated from all RNA samples. Gene-specific primers were used to amplify target sequences using AmpliTaq Gold 360 Core Mixture (Thermo Fisher-Applied Biosystems, Waltham, MA, USA). PCR reactions were visualized on 2% agarose gels. A positive control primer set amplifying a constitutively expressed housekeeping gene (Actb) encoding a cell cytoskeletal protein was included. Results are shown in Figure 2. Pluripotency markers Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1 were detected by rt-PCR from miPSC cells, but not from parental fibroblasts.

[0214] Pluripotency was also tested by immunofluorescence. miPSCs were plated in 24-well plates and processed for RT-PCR and immunocytochemistry (ICC) analysis 48 h after plating. For ICC, cells were fixed, permeabilized, and blocked using the Image-iT Fixation / Permeabilization Kit (Thermo Fisher Scientific, Petition 870240080912, dated 09 / 23 / 2024, page 85 / 136 79 / 105 Waltham, MA, USA). Cells were stained overnight at 4°C with primary antibodies to Sox2 and Oct4. After several washes, the cells were incubated with AlexaFluor 488 secondary antibody and NucBlue Fixed Cell ReadyProbes reagent (Thermo Fisher Scientific). Stained cells were visualized using a fluorescence microscope and were positive for Sox2 and Oct4. Data not shown.

[0215] Figure 3 further confirms pluripotency by a functional assay. 2 x 106 miPSC cells were injected into the thigh muscle of recipient C57BL / 6 mice (syngeal), BALB / c mice (allogeneic), nude BALB / c mice (allogeneic but not T cell deficient), and beige SCID mice (immunodeficient). Teratomas were formed in all mice except the immunocompetent allogeneic BALB / c mice.

[0216] β-2 Microglobulin Knockout: CRISPR technology was used for knockout of the B2m gene. To target the coding sequence of the mouse β-2 microglobulin (B2m) gene, the CRISPR sequence 5'TTCGGCTTCCCATTCTCCGG (TGG)-3' was paired and ligated into All-In-One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher Scientific, Waltham, MA, USA). (Other CRISPRs that worked, but were less effective, were 5'-GTATACTCACGCCACCCAC (CGG)-3' and 5'-GGCGTATGTATCAGTCTCAG (TGG)-3'). miPSCs were transfected with the AIO vectors using Neon electroporation with two 1200 V pulses of 20 ms duration. The transfected iPSC cultures were dissociated into single cells using 0.05% Trypsin (Gibco) and then classified using the classifier of Petition 870240080912, dated 09 / 23 / 2024, page 86 / 136 80 / 105 FACSAria™ cells (BD Bioscience, Franklin Lakes, NJ, USA) were used to remove doublets and debris by selective selection in forward and side scatter emission. Individual cells were expanded into full-size colonies and tested for CRISPR edits by scanning for the presence of the aberrant CRISPR cleavage site sequence. Briefly, the target sequence was amplified via PCR using AmpliTaq Gold Mastermix (Thermo Fisher-Applied Biosystems, Waltham, MA, USA) and the B2m gDNA primers.

[0217] F: 5'- CTGGATCAGACATATGTGTTGGGA-3',

[0218] R: 5'-GCAAAGCAGTTTTAAGTCCACACAG-3'

[0219] After cleaning the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher Scientific, Waltham, MA, USA), Sanger sequencing was performed using an Ion Personal Genome Machine (PGM™, Thermo Fisher Scientific). Sequencing to identify homogeneity, a 250 bp region of the B2m gene, was amplified by PCR using B2m gDNA PGM primers:

[0220] F: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3' and

[0221] R: 5' - GGATTTCAATGTGAGGCGGGT-3'.

[0222] The PCR product was purified as previously described and prepared using the Ion PGM Hi-Q Model Kit (Thermo Fisher Scientific). Experiments were performed on the Ion PGM™ System with the Ion 318™ Chip Kit (Thermo Fisher Scientific). Pluripotency analyses were performed again.

[0223] As seen in Figure 4, β2-microglobulin expression was eliminated in miPSC cells. MHC-I expression was not induced by IFN-γ stimulation (panel Petition 870240080912, dated 09 / 23 / 2024, page 87 / 136 81 / 105 from the right). As a control, parental miPSC cells were stimulated with IFN-γ (left panel). c. EXAMPLE 2: GENERATION OF PLURIPOTENT CELLS SUBMITTED TO DOUBLE KNOCKOUT β-2 MICROGLOBULIN / CIITA

[0224] CRISPR technology was used for additional knockout of the Ciita gene. To target the mouse Ciita gene coding sequence, the CRISPR sequence 5'-GGTCCATCTGGTCATAGAGG (CGG)-3' was paired and ligated into All-In-One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher, Waltham, MA, USA). miPSCs were transfected with the AIO vectors using the same conditions as for B2m-KO. Transfected iPSC cultures were dissociated into individual cells using 0.05% Trypsin (Thermo Fisher-Gibco) and then separated with the FACSAria™ cell separator (BD Bioscience, Franklin Lakes, NJ, USA) to remove doublets and debris by selective sorting in forward and side scatter emission. Individual cells were expanded into full-size colonies and tested for CRISPR edits by scanning for the presence of the aberrant CRISPR cleavage site sequence.In summary, the target sequence was amplified via PCR using AmpliTaq. Gold Mastermix (Thermo Fisher Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA primers F: 5'-CCCCCAGAACGATGAGCTT3', R: 5'-TGCAGAAGTCCTGAGAAGGCC-3'. After cleaning the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher, Waltham, MA, USA), Sanger sequencing was performed. Using the DNA sequence chromatogram, the edited clones were then identified through the presence of the aberrant CRISPR cleavage site sequence. The size Petition 870240080912, dated 09 / 23 / 2024, page 88 / 136 An indel value of 82 / 105 was calculated using the TIDE tool. PCR and ICC were performed again to verify the pluripotency status of the cells.

[0225] Figure 5 confirms the double knockout of miPSC / B-2-microglobulin / Ciita. MHC-II cannot be induced by TNF-α to express MHC-II. D. EXAMPLE 3: GENERATION OF DOUBLE KNOCKOUT β-2 MICROGLOBULIN / CYITA CD47+ PLURIPOTENT CELLS

[0226] A Cd47 expression vector was introduced into the B2m / Ciita double-knockout miPSC generated above. The vector was delivered using lentivirus containing the Blasticidin antibiotic resistance cassette. The Cd47 gene sequence was synthesized and the DNA was cloned into the pLentivirus Lentivirus plasmid (ThermoFisher, Waltham, MA, USA) containing a blasticidin resistance marker. Sanger sequencing was performed to verify that no mutations had occurred. Lentivirus generation was performed with a stock titer of 1 x 10⁷ TU / ml. The recombinant vector was transduced into 2 x 10⁵ B2M-KO / CIITA double-knockout mIPSCs, cultured in blasticidin-resistant MEF cells for 72 h with a MOI ratio of 1:10 followed by antibiotic selection with 12.5 µg / ml of blasticidin for 7 days. The selected antibiotic banks were tested by RT-qPCR amplification of Cd47 mRNA and detection by flow cytometry of Cd47. After confirmation of Cd47 expression, the cells were expanded and subjected to pluripotency assays.

[0227] Figure 6A shows the increased expression of Cd47 from a transgene added to the double knockout β2-microglobulin / Ciita (iPShipo cells). Figure 6B shows that C57BL / 6 iPS hypo cells survive in the environment. Petition 870240080912, dated 09 / 23 / 2024, page 89 / 136 83 / 105 allogeneic BALB / c cells, but the parental iPS cells did not. This new result confirms that hypoimmune pluripotent cells survive when transplanted into incompatible hosts. E. Differentiation of mouse cells from MHIP cells

[0228] Islet cells: mHIP cells were differentiated into islet cells using techniques adapted from Liu et al., Exp. Diabetes Res 2012: 201295 (doi:10.1155 / 2012 / 201295), incorporated herein by reference and in particular for the differentiation techniques presented therein. iPS cells were transferred to gelatin-coated flasks for 30 min to remove the feeder layer and seeded at 1 x 10⁶ cells per well onto collagen I-coated plates in DMEM / F-12 medium supplemented with 2 mM glutamine, 100 μM non-essential amino acid, 10 ng / ml activin A, 10 mM nicotinamide and 1 μg / ml laminin with 10% FBS overnight. ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 μM non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, 25 pg / ml insulin, and 1 pg / ml laminin with 2% FBS for 6 days.

[0229] Neural stem cells: mHIP cells were differentiated into neural cells using techniques adapted from Abraches et al., doi:10.1371 / journal.pone.0006286, incorporated herein by reference and in particular for the differentiation techniques presented therein. To initiate the monolayer protocol, ES cells were plated on serum-free ESGRO Complete Clonal Grade medium (Millipore) at high density (1.5 χ¹⁰⁵ cells / cm²). After 24 hours, the ES cells were gently dissociated and Petition 870240080912, dated 09 / 23 / 2024, pp. 90 / 136 84 / 105 cells were plated onto 0.1% (v / v) gelatin-coated tissue culture plastic at 1 x 10⁴ cells / cm² in RHB-A or N2B27 medium (StemCell Science Inc.), changing the medium between each day. For replating on day 4, the cells were dissociated and plated at 2 x 10⁴ cells / cm² on laminin-coated tissue culture plastic in medium supplemented with RHB-A with 5 ng / ml of murine bFGF (Peprotech). From this point, the cells were replated under the same conditions every 4 days and the medium was changed every 2 days, totaling 20 days in culture. To quantify the number of differentiating neurons at each point in time, cells were plated on laminin-coated glass coverslips in 24-well, Nunc plates, and 2 days after plating, the medium was changed to a mixture of RHB-A:Neurobasal:B27 (1:1:0.02) to allow for better survival of differentiated neurons.

[0230] Smooth muscle cells: mHIP cells were differentiated into SM cells using techniques adapted from Huang et al., Biochem Biophys Res Commun 2006: 351 (2) 321-7, incorporated herein by reference and in particular for the differentiation techniques described therein. Resuspended iPSCs were cultured in 6-well gelatin-coated plastic petri dishes (Falcon, Becton-Dickinson) at 2 million cells per well at 37 °C, 5% CO2 in 2 ml of differentiation medium with the presence of 10 μM atRA, respectively. The differentiation medium was produced from DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin, and streptomycin. Culture was continued for 10 days with daily exchange of fresh media.

[0231] From the 11th day onwards, the middle of Petition 870240080912, dated 09 / 23 / 2024, pp. 91 / 136 85 / 105 differentiation was replaced with serum-free culture medium, composed of knockout DMEM, 15% knockout serum replacement, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin, and streptomycin. Cultures were continued for a further 10 days with daily changes of the serum-free medium.

[0232] Cardiomyocytes: mHIP cells were differentiated into CM cells using techniques adapted from Kattman et al., Cell Stem Cell 8: 228-240 (2011), incorporated here by reference and in particular for the differentiation techniques presented therein.

[0233] Endothelial cells: mHIP cells have differentiated into endothelial cells, as is known. F. EXAMPLE 4: ALLOGENEIC TRANSPLANTATION OF HIP CELL DERIVATIVES SHOWS LONG-TERM SURVIVAL IN FULLY IMMUNOCOMPETENT RECIPIENTS a. MICE:

[0234] BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU, CAnN.CgFoxn1 / / Crl, H2d) and beige Scid (CBySmn. CB17-Prkdcscid / J) (all 6-12 weeks) were used as recipients for different assays (all 6-12 weeks of age). The number of animals per experimental group is shown in each Figure. Mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and received humane care in accordance with the Guide to the Principles of Laboratory Animals. Animal experiments were approved by the Hamburg Office for Health and Consumer Protection and conducted in accordance with local and EU guidelines. b. PLURIPOTENCE ANALYSIS BY RT-PCR AND IF: Petition 870240080912, dated 09 / 23 / 2024, page 92 / 136 86 / 105

[0235] miPSCs were plated in 24-well plates and processed for RT-PCR and immunofluorescence (IF) 48 h post-plating. For ICC, cells were fixed, permeabilized, and blocked using the Kit of Image-iT™ Fixation / Permeabilization (Thermo Fisher Cat. No., (R37602). The cells were stained overnight at 4°C with primary antibodies to Sox2, SSEA-1, Oct4, and Alkaline Phosphatase. After several washes, the cells were incubated with AlexaFluor 488 secondary antibody and NucBlue Fixed Cell ReadyProbes reagent (all Thermo Fisher Scientific). The stained cells were photographed using a fluorescence microscope.

[0236] For RT-PCR, RNA was extracted using the Mini RNA PureLink™ Kit (Thermo Fisher Cat. No. 12183018A). A DNase I step was included to remove contaminating genomic DNA. cDNA was generated using the Applied Biosystems® High-Power cDNA Reverse Transcription Kit. Non-reverse transcriptase (no RT) controls were also generated from all RNA samples. Gene-specific primers were used to amplify target sequences using AmpliTaq Gol® 360 Master Mix (Thermo Fisher Cat. No. 4398876). PCR reactions were visualized on 2% agarose gels. A set of positive control primers amplifying a constitutively expressed housekeeping gene (Actb) encoding a cell cytoskeletal protein was included. c. GENETIC EDITION OF MOUSE IPSCS:

[0237] miPSCs were subjected to 3 gene editing steps. First, CRISPRs targeting the mouse B2m gene coding sequence were Petition 870240080912, dated 09 / 23 / 2024, pp. 93 / 136 87 / 105 paired and ligated in vectors containing the Cas9 expression cassette. The transfected miPSCs were dissociated into individual cells, expanded into colonies, sequenced, and tested for homogeneity. Secondly, these B2m- / - miPSCs were transfected with vectors containing CRISPRs targeting Ciita, the major regulator of MHC II molecules. The expanded individual cell colonies were sequenced, and the B2m- / - Ciita- / - clones were identified through the presence of an aberrant CRISPR cleavage site sequence. Thirdly, the Cd47 gene sequence was synthesized, and the DNA was cloned into a plasmid lentivirus with blasticidin resistance. The B2m- / - Ciita- / - miPSCs were transfected and cultured in the presence of blasticidin. Selected antibiotic clusters were tested for Cd47 overexpression, and B2m- / -Ciita- / -Cd47tg miPSCs were expanded.FACS analyses demonstrated elevated MHC I expression, modest but detectable MHC II expression, and negligible Cd47 expression in wt miPSCs. The lack of MHC I expression, MHC II expression, and Cd47 overexpression in the engineered miPSC lines was confirmed. All engineered miPSC lines were tested for pluripotency. This was confirmed in B2m- / Ciita- / -Cd47tg miPSCs after 3 manipulation steps and their potential to form cells from all 3 germ layers. d. Generation of MIPSCS B2M- / -:

[0238] CRISPR technology was used for B2m gene knockout. To target the coding sequence of the mouse beta-2-microglobulin (B2m) gene, the CRISPR sequence 5'-TTCGGCTTCCCATTCTCCGG (TGG)-3' was paired and ligated into All-In-One (AIO) vectors containing the Petition 870240080912, dated 09 / 23 / 2024, pp. 94 / 136 88 / 105 Cas9 expression cassettes were transfected according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher, Waltham, MA, USA). miPSCs were transfected with AIO vectors using neon electroporation with two 1200 V pulses of 20 ms duration. Transfected iPSC cultures were dissociated into single cells using 0.05% trypsin (Gibco) and then sorted with the FACSAria cell sorter (BD Bioscience, Franklin Lakes, NJ) to remove doublets and debris by selective sorting in forward and side scatter emission. Individual cells were expanded to full-size colonies and tested for CRISPR editing by scanning for the presence of aberrant CRISPR cleavage site sequences.In summary, the target sequence was amplified by PCR using AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and the primers B2M ADNg F: 5'CTGGATCAGACATATGTGTTGGGA-3', R: 5'-GCAAAGCAGTTTTAAGTCCACACAG3'. After cleaning the obtained PCR product (PureLink® Pro 96). Sanger sequencing was performed using a PCR Purification Kit (Thermo Fisher). For homogeneity identification, a 250 bp region of the B2m gene was amplified by PCR using B2m gDNA primers. PGM F: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3' and R: 5'GGATTTCAATGTGAGGCGGGT-3'. The PCR product was purified as previously described and prepared using the Ion PGM Hi-Q Model Kit (Thermo Fisher). The experiments were performed on Ion PGM™ System with the Ion 318™ Chip Kit v2 (Thermo Fisher). Pluripotency analyses were performed again.

[0239] A reduced growth rate or differentiation capacity of B2m- / - iPSCs was not observed. Petition 870240080912, dated 09 / 23 / 2024, pp. 95-136 89 / 105 as previously reported in the technique. e. Generation of MIPSCS B2M- / - and CIITA- / -:

[0240] CRISPR technology was used for additional knockout of the Ciita gene. To target the mouse Ciita gene coding sequence, the CRISPR sequence 5'-GGTCCATCTGGTCATAGAGG (CGG)-3' was paired and ligated into All-In-One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher, Waltham, MA, USA). miPSCs were transfected with AIO vectors using the same conditions for B2m-KO. Transfected miPSC cultures were dissociated into individual cells using 0.05% Trypsin (Gibco) and then separated with FACSAria cell sorter (BD). Bioscience, Franklin Lakes, NJ) to remove doublets and debris by selective sorting in forward and side scatter emission. Individual cells were expanded into full-size colonies and tested for CRISPR editing by screening for the presence of aberrant CRISPR cleavage site sequences. Briefly, the target sequence was amplified via PCR using AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA primers F: 5'-CCCCCAGAACGATGAGCTT-3', R: 5'TGCAGAAGTCCTGAGAAGGCC-3'. After cleaning the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. Using the DNA sequence chromatogram, the edited clones were then identified by the presence of an aberrant CRISPR cleavage site sequence. The indel size was calculated using the TIDE tool. PCR and ICC were performed again to verify pluripotency status. Petition 870240080912, dated 09 / 23 / 2024, pp. 96 / 136 90 / 105 of the cells. f. Generation of MIPSCS B2M- / - CIITA- / - CD47TG:

[0241] The iPSC B2m-KO, Ciita-KO and cellular line Cd47-tg was generated through antibiotic resistance selection following lentivirus-mediated administration of a Cd47 expression vector containing the Blasticidin antibiotic resistance cassette. The Cd47 gene sequence was synthesized and the DNA was cloned into the pLentivirus pLenti6 / V5 (ThermoFisher) plasmid with blasticidin resistance. Sanger sequencing was performed to verify that no mutations occurred. Lentivirus generation was performed with a stock titer of 1 x 10⁷ TU / ml. Transduction was performed in 2 x 10⁵ miPSCs B2m- / - Ciitas- / -, cultured in blasticidin-resistant MEF cells for 72 h with a MOI ratio of 1:10 followed by antibiotic selection with 12.5 μg / ml of Blasticidin for 7 days. Selected antibiotic-treated cell banks were tested by RT-qPCR amplification of Cd47 mRNA and flow cytometry detection of Cd47. After Cd47 confirmation, the cells were expanded and confirmed by pluripotency tests. g. Derivation and characterization of iIPSC-derived endothelial cells (iECs):

[0242] iECs were derived using a three-dimensional approach. Briefly, to initiate differentiation, iPSCs were cultured on ultralow non-adhesive plates to form embryonic body (EB) aggregates in EBM2 medium (Lonza) in the absence of leukemia inhibitory factor (LIF). After 4 days of suspension culture, the EBs were re-fixed onto 0.2% gelatin-coated plates and cultured in EBM2 medium supplemented with VEGF-A165 (50 ng / ml; Petition 870240080912, dated 09 / 23 / 2024, pp. 97 / 136 91 / 105 (PeproTech). After 3 weeks of differentiation, individual cell suspensions were obtained using a cell dissociation buffer (Life Technologies) and labeled with APC-conjugated CD31 anti-mouse antibodies (eBiosciences) and PE-conjugated CD144 antibodies (BD Biosciences). iECs were purified by fluorescence-activated cell screening (FACS) of the CD31+CD144+ population. iECs were maintained in EBM2 medium supplemented with recombinant murine vascular endothelial growth factor (50 ng / ml).

[0243] Its phenotype was confirmed by immunofluorescence for CD31 and VE cadherin, as well as by PCR and tube formation assays to demonstrate endothelial function in forming premature vascular structures. Note: Differentiation protocols using confluent iPSC monolayers in 0.1% gelatin or Matrigel were also successful. Note: Other endothelial cell media were also used successfully. h. Derivation and Characterization of iPSC-Derived Smooth Muscle Cells (iSMCs):

[0244] Resuspended iPSCs were cultured in 6-well Petri dishes coated with 0.1% gelatin (Falcon, Becton-Dickinson) at 2 million cells per well at 37 °C, 5% CO2 in 2 ml of differentiation medium containing 10 µM. The differentiation medium was made from DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin, and streptomycin. The culture was continued for 10 days with daily medium changes.

[0245] From day 11 onwards, the differentiation medium was replaced with a culture medium free of Petition 870240080912, dated 09 / 23 / 2024, pp. 98 / 136 92 / 105 serum from a knockout DMEM: replacement of knockout serum at 15%, L-glutamine 2 mM, MTG 1 mM, non-essential amino acids 1%, penicillin, and streptomycin. Cultures were continued for a further 10 days with daily changes in serum-free medium. The phenotype was confirmed by immunofluorescence and PCR for both SMA and SM22. i. Derivation and Characterization of iPSC-Derived Cardiomyocytes (iCMs):

[0246] Prior to differentiation, iPSCs were passed twice through gelatin-coated plates to remove feeder cells. Briefly, iPSCs were dissociated with TrypLE (Invitrogen) and cultured at 75,000 to 100,000 cells / ml without any additional growth factors for 48 hours. Three-day-old EBs were dissociated and the cells were differentiated into “cardiac conditions”. In summary, 6 χ¹⁰⁴ to 10 χ¹⁰⁴ cells were seeded into individual wells of a 96-well flat-bottom plate (Becton Dickenson, Franklin Lakes, NJ, USA) coated with gelatin in StemPro-34 SF medium (Invitrogen), supplemented with 2 mM L-glutamine, 1 mM ascorbic acid (Sigma), human VEGF (5 ng / ml), human DKK1 (150 ng / ml), human bFGF (10 ng / ml), and human FGF10 (12.5 ng / ml) (R&D Systems). Cultures were harvested 4 or 5 days later (total of 7 or 8 days).

[0247] Its phenotype was confirmed by IF for troponin I and sarcomeric alpha-actinin, as well as PCR for Gata4 and Mhy6. The cells began beating between 8-10 days. This demonstrated their functional differentiation. j. Derivation and Characterization of Islet Cells Derived from iPSCs (iICs)

[0248] The iPS cells were transferred to Petition 870240080912, dated 09 / 23 / 2024, pp. 99 / 136 93 / 105 flasks were gelatin-coated for 30 min to remove the feeder layer and seeded at 1 x 106 cells per well onto collagen-I coated plates in DMEM / F-12 medium supplemented with 2 mM glutamine, 100 μM non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, and 1 μg / ml laminin with 10% FBS overnight. ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 μM non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, 25 pg / ml insulin, and 1 pg / ml laminin with 2% FBS for 6 days. Its phenotype was confirmed by immunofluorescence for C-peptide, PCR for glucagon, Ngn3, amylase, insulin 2, somatostatin, and insulin production. k. Derivation and Characterization of iPSC-Derived Neuronal Cells (iNCs)

[0249] To initiate the monolayer protocol, iPSCs were gently dissociated and plated onto 0.1% gelatin-coated culture plastic tissue at 1 x 10⁴ cells / cm² in RHB-A or N2B27 media (StemCell Science Inc.), changing the medium every two days. For replating on day 4, cells were dissociated and plated at 2 x 10⁴ cells / cm² onto laminin-coated culture plastic tissue in RHB-A medium supplemented with 5 ng / ml murine bFGF (Peprotech). From this point, cells were replated under the same conditions every 4 days, and the medium was changed every 2 days, totaling 20 days in culture. To quantify the number of differentiating neurons at each point in time, cells were plated onto laminin-coated glass coverslips in 24-well Nunc plates, and 2 days after plating, the medium was changed to a mixture of Petition 870240080912, dated 09 / 23 / 2024, pp. 100 / 136 94 / 105 RHB-A:Neurobasal:B27 (1:1:0.02), to allow for better survival of differentiated neurons. Its phenotype was confirmed by IF for Tuj-1 and nestin. 1. ELISPOT ESSAYS

[0250] For the unidirectional EnmuneLinked ImmunoSpot (ELISPOT) assays, recipient splenocytes were isolated from fresh spleen 5 days post-cell injection (miPSC, miPSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m- / - Ciita- / - Cd47tg) and used as responder cells. Donor cells (miPSC, miPSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m- / - Ciita- / - Cd47tg) were inhibited by mitomycin and served as stimulator cells. 10⁶ stimulator cells were incubated with 5x10⁵ recipient responder splenocytes for 24 hours. Spot frequencies of IFNγ and IL-4 were automatically enumerated using an ELISPOT plate reader. Quadruplicates were performed in all assays. m. Teratoma assays to study the survival of Ipsc in vivo

[0251] Syngeneic or allogeneic mice aged six weeks were used for transplantation of non-immunogenic wtiPSCs or iPSCs. 1x106 cells were injected in 100 µl into the right thigh muscle of mice. Transplanted animals were routinely observed every two days, and tumor growth was measured with a caliper. They were sacrificed after the development of tumors larger than 1.5 cm3 or after an observation period of 100 days. n. In vitro NK cell assays

[0252] The expression of CD107 in NK cells after co-culture with wtiPSCs or with HIP cells was measured by Petition 870240080912, dated 09 / 23 / 2024, pp. 101 / 136 95 / 105 flow cytometry as a marker of NK cell activation. Using the Elispot principle, NK cells were co-cultured with wtiPSCs or HIP cells and their IFN-γ release was measured.

[0253] According to the “self” theory, it has been demonstrated that MHC I-deficient stem cells are susceptible to NK cell killing, since both murine and human PSCs express ligands to activate NK receptors. Although the expression of activating receptors has been reported to decrease with differentiation, NK cell killing of B2m- / - derivatives has been observed. While isolated expression of HLAE or HLA-G in human pluripotent stem cells has been used to mitigate the expected innate immune response in HLA I- / - cells, there are additional highly effective non-MHC inhibitory ligands among them. The invention reports that Cd47 has been found to be a surprisingly potent inhibitor of innate immune clearance. o. SUMMARY OF MOUSE DATA

[0254] All engineered miPSC lines were transplanted into syngeneic and allogeneic BALB / c C57BL / 6 receptors without any immunosuppression. Although all engineered cells similarly developed teratomas in syngeneic receptors, their survival depended on their level of hypoimmunogenicity in allogeneic receptors. A 60% teratoma formation was observed in B2m- / - miPSCs in BALB / c, along with a subtle Elispot response and a measurable IgM antibody response. In B2m- / - Ciita- / - miPSCs, a 91.7% teratoma formation was observed in allogeneic BALB / c, a smaller Elispot response, and no antibody response. The final B2m- / - Ciita- / - Cd47tg miPSC line showed 100% teratoma formation. Petition 870240080912, dated 09 / 23 / 2024, pp. 102 / 136 96 / 105 teratoma and no Elispot or antibody response. The contribution of CD47 overexpression was further evaluated in innate immunity assays, comparing B2m- / -Ciita- / - miPSCs with B2m- / -Ciita- / -Cd47tg miPSCs. CD47 overexpression significantly reduced CD107 expression from NK cells and IFN-γ release from NK cells, thus mitigating innate immune clearance. In summary, each step of the manipulation made the miPSCs more hypoimmunogenic.

[0255] B2m- / -Ciita- / -HIP cells differentiated into hypoimmunogenic endothelial-like cells (miECs), smooth muscle-like cells (miSMCs), and cardiomyocyte-like cells (miCMs). “Wild-type” miPSC derivatives (i.e., from unmanipulated miPSCs) served as controls. All derivatives showed the typical morphological appearance, cell marker immunofluorescence, and gene expression of their intended mature tissue cell lines. The expression of MHC I and II molecules in wt derivatives was generally largely upregulated compared to their parental miPSC line, but markedly varied by cell type. As expected, miECs had by far the highest expression of MHC I and MHC II, miSMCs had moderate expression of MHC I and MHC II, while miCMs had moderate expression of MHC I but very low expression of MHC II.All wt derivatives had a fairly low Cd47 expression, although also slightly higher than that of miPSCs. All B2m- / -Ciita- / -Cd47tg derivatives appropriately showed a complete lack of MHC I and MHC II. II and Cd47 are significantly larger than their wt counterparts.

[0256] Matrigel plugs containing 5x105mi miECs Petition 870240080912, dated 09 / 23 / 2024, pp. 103 / 136 97 / 105 wt, miSMCs, and miCMs were transplanted into subcutaneous pockets of syngeneic C57BL / 6 or allogeneic BALB / c mice. After 5 days, all allogeneic recipients mounted a strong cellular immune response, as well as a strong IgM antibody response against these wild-type cell grafts. In marked contrast, none of the corresponding B2m- / Ciita- / -Cd47tg (HIP) derivatives showed detectable increases in IFN-γ Elispot frequencies or IgM antibody production.

[0257] The morphology of the transplanted cells was also confirmed. Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 mi cells, MIPSC-derived cardiomyocytes (miCM), MIPSC-derived smooth muscle cells (miSMC), or miPSC-derived endothelial cells (miEC) in 250 µl of 0.9% saline solution were mixed with 250 µl of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding. 5 µm thick sections were cut and stained with hematoxylin and eosin (H&E). Histology confirmed morphologically adequate miCMs, miSMCs, and miECs. g. EXAMPLE 5: GENERATION OF HUMAN IPCS

[0258] The Human Episomal iPSC Cell Line was derived from CD34+ cord blood (Cat. No. A33124, Thermo Fisher) Scientific) using a three-plasmid, seven-factor EBNA-based episomal system (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, Petition 870240080912, dated 09 / 23 / 2024, pp. 104 / 136 98 / 105 The iPSC line is composed of MYC, NANOG, LIN28, and SV40L T antigen) from ThermoFisher. This iPSC line is considered to have a zero footprint due to the fact that there was no integration into the genome from the reprogramming event. It has been shown to be free of all reprogramming genes. The iPSCs have a normal XX karyotype and endogenous expression of pluripotent markers such as Oct4, Sox2, Nanog (as shown by RT-PCR), Oct4, SSEA4, TRA-1-60, and TRA-1-81 (as shown by ICC). In directed differentiation and teratoma analyses, these hiPSCs maintained their differentiation potential for ectodermal, endodermal, and mesodermal lineages. Furthermore, vascular, endothelial, and cardiac lineages were derived with robust efficiencies.

[0259] Note: several gene delivery vehicles for iPSC generation have been used successfully, including retroviral vectors, adenoviral vectors, Sendai virus, as well as virus-free reprogramming methods (using episomal vectors, piggyBac transposon, synthetic mRNAs, microRNAs, recombinant proteins and small molecule drugs, etc.).

[0260] Note: Different factors have been used successfully for reprogramming, such as the first reported combination of OCT3 / 4, SOX2, KLF4, and C-MYC, known as Yamanaka factors. In one embodiment, only three of these factors were successfully combined and C-MYC omitted, although with reduced reprogramming efficiency.

[0261] In one embodiment, L-MYC or GLIS1 instead of C-MYC showed better reprogramming efficiency. In another embodiment, the reprogramming factors are not limited to genes associated with pluripotency. Petition 870240080912, dated 09 / 23 / 2024, pp. 105 / 136 99 / 105 a. STATISTICS

[0262] All data are expressed as mean ± SD or in box plots with the median and the minimum-to-maximum range. Intergroup differences were adequately assessed by unpaired Student's t-test or by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. * p<0.05, ** p<0.01. H. EXAMPLE 6: GENERATION OF HUMAN HIP CELLS

[0263] Human IPSC Cas9 cells underwent two gene editing steps. In the first step, CRISPR technology was performed by a combined targeting of the human beta-2-microglobulin (B2M) gene coding sequence with the CRISPR sequence 5'-CGTGAGTAAACCTGAATCTT-3' and the human CIITA gene coding sequence with the CRISPR sequence 5'GATATTGGCATAAGCCTCCC-3' linearized with the T7 promoter to synthesize gRNA according to the kit instructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). The collected in vitro transcribed gRNA (IVT) was then purified using the MEGAclear Transcription Cleanup Kit. For IVT RNA administration, singularized cells were electroporated with 300 ng of IVT gRNA using a Neon electroporation system.Following electroporation, edited Cas9 iPSCs were expanded for single-cell inoculation: iPSC cultures were dissociated into isolated cells using TrypLE (Gibco) and stained with Alexa Fluor 488 Tra1-60 and propidium iodide (PI). The FACS Aria cell sorter (BD Biosciences) was used for sorting, and doublets and debris were excluded from seeding by selective sorting in forward and side scattering emissions. Viable pluripotent cells were selected. Petition 870240080912, dated 09 / 23 / 2024, pp. 106 / 136 100 / 105 cells were selected in the absence of PI and presence of Tra1-60 Alexa Fluor 488 staining. Individual cells were then expanded into full-size colonies, after which the colonies were tested for CRISPR editing. CRISPR-mediated cleavage was assessed using the GeneArt Genomic Cleavage Detection Kit (Thermo Fisher). Genomic DNA was isolated from 1x10⁶ hiPSCs and CIITA genomic DNA regions of B2M and amplified by PCR using AmpliTaq Gold 360 Master Mix and primer sets F: 5'TGGGGCCAAATCATGTAGACTC-3' and R: 5'-TCAGTGGGGGTGAATTCAGTGT-3' for B2M as well as F: 5'-CTTAACAGCGATGCTGACCCC-3' and R: 5'TGGCCTCCATCTCCCCTCTCTT-3' for CIITA. For TIDE analysis, the PCR product obtained was cleaned (PureLink PCR Purification Kit, Thermo Fisher) and Sanger sequencing was performed for indel frequency prediction.After confirmation of the B2M / CIITA knockout, the cells were further characterized through karyotype analysis and the TaqMan hPSC Classification Panel (Thermo Fisher). The PSC was considered pluripotent and maintained a normal karyotype (46, XX) during the genome editing process.

[0264] In the second stage, the CD47 gene was synthesized and the DNA was cloned into a plasmid lentivirus with an EF1a promoter and resistance to puromycin. Cells were transduced with lentiviral stocks of 1x107 TU / ml and 6 μg / ml of Polybrene (Thermo Fisher). The medium was changed daily after transduction. Three days after transduction, the cells were expanded and selected with 0.5 μg / ml of puromycin. After 5 days of antibiotic selection, antibiotic-resistant colonies appeared and were expanded to generate stable clusters. The CD47 level was confirmed by qPCR. Petition 870240080912, dated 09 / 23 / 2024, pp. 107 / 136 101 / 105 Pluripotency assay (TaqMan hPSC Classification Panel, Thermo Fisher) and karyotyping were performed again to verify the pluripotent state of the cells. I. EXAMPLE 7: DIFFERENTIATION OF HUMAN HIP CELLS 1. Differentiation of hHIP cells from human cardiomyocytes

[0265] This was performed using a protocol adapted from Sharma et al., J. Vis Exp. 2015 doi:10.3791 / 52628, incorporated herein by reference in its entirety and specifically for the techniques to differentiate the cells. hiPSCs were plated on diluted Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8 Flex medium (Thermo Fisher). After the cells reached 90% confluence, differentiation was initiated and the medium was changed to 5 ml of RPMI1640 containing B-27 at 2% less Insulin (both Gibco) and CHIR-99021 6 uM (Selleck Chem). After 2 days, the medium was changed to RPMI1640 containing B-27 at 2% less Insulin without CHIR. On day 3, 5 µl of IWR1 were added to the medium for two more days. On day 5, the medium was changed back to RPMI 1640 containing B-27 medium with 2% less insulin and incubated for 48 h. On day 7, the medium was changed to RPMI 1640 containing B27 plus insulin (Gibco) and replaced every 3 days thereafter with the same medium.Spontaneous cardiomyocyte beating was first visible approximately between days 10 and 12. Cardiomyocyte purification was performed on day 10 after differentiation. Briefly, the medium was changed to low-glucose medium and maintained for 3 days. On day 13, the medium was changed back to RPMI 1640 containing B27 plus insulin. This procedure was repeated on day 14. The remaining cells are highly purified cardiomyocytes. Petition 870240080912, dated 09 / 23 / 2024, pp. 108 / 136 102 / 105 2. Differentiation of hHIP cells from human endothelial cells

[0266] HiPSCs were plated on diluted Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8 Flex medium (Thermo Fisher). After the cells reached 60% confluence, differentiation was initiated and the medium was changed to RPMI1640 containing B-27 at 2% less insulin (both from Gibco) and CHIR-99021 5 μM (Selleck Chem). On day 2, the medium was changed to reduced medium: RPMI1640 containing B-27 at 2% less insulin (both from Gibco) and CHIR-99021 2 μM (Selleck Chem). From day 4 to day 7, cells were exposed to RPMI EC medium, RPMI1640 containing B-27 at 2% less Insulin plus 50 ng / ml vascular endothelial growth factor (VEGF; R & D Systems, Minneapolis, MN, USA), 10 ng / ml basic fibroblast growth factor (FGFb; R & D Systems), Y27632 10 μM (Sigma-Aldrich, Saint Louis, MO, USA) and SB 431542 μM (Sigma-Aldrich).Endothelial cell clusters were visible from day 7 and the cells were maintained in EGM-2 SingleQuots medium (Lonza, Basel, Switzerland) plus 10% FCS hi (Gibco), 25 ng / ml vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 2 ng / ml basic fibroblast growth factor (FGFb; R&D Systems), Y-27632 10 μM (Sigma-Aldrich, Saint Louis, MO, USA) and SB 431542 1 μM (Sigma-Aldrich). The differentiation process was completed after 14 days and undifferentiated cells were detached during the differentiation process. For purification, the cells underwent MACS processing according to the manufacturer's protocol using CD31 microspheres (Miltenyi, Auburn, CA). Highly purified EC cells were cultured in EGM-2 SingleQuots medium (Lonza, Basel, Switzerland). Petition 870240080912, dated 09 / 23 / 2024, pp. 109 / 136 103 / 105 plus supplements and FCS hi at 10% (Gibco). TrypLE was used to pass the cells 1:3 every 3 to 4 days. j. TRANSPLANTATION IN HUMANIZED MICE

[0267] Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1. Myo cells, or hiPSC-derived cardiomyocytes (hiCM) or hiPSC-derived endothelial cells (hiECin), 250 µl of 0.9% saline solution containing ZVAD (100 mM, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, Calbiochem), Bcl-XL BH4 (cell-permeated TAT peptide, 50 nM, Calbiochem), cyclosporine A (200 nM, Sigma), IGF-1 (100 ng / ml, Peprotech), and pinacidil (50 mM, Sigma) were mixed with 250 µl of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower dorsal region of mice using a 23-G syringe. After implantation, they were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding.A 5 µm thick section was cut and stained with Hematoxylin and Eosin (HE), and the morphology was confirmed. ix. EXEMPLARY SEQUENCES: SEQ ID NO:- 1 Human β-2-Microglobulin

[0268] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSN FLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRV NHVTLSQPKIVKWDRDI SEQ ID NO:2 - Human CIITA protein, N-terminal 160 amino acids

[0269] MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSD ADPLCLYHFYDQMDLAGEEEIEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELD Petition 870240080912, dated 09 / 23 / 2024, pp. 110 / 136 104 / 105 QYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKP SEQ ID NO: 3 - Human CD47

[0270] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPC FVTNMEAAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLH YYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLLSILALAQLLG LVYMKFVE SEQ ID NO: 4 - Timidina Quinase do Virus do Herpes Simplex (HSV-tk)

[0271] MASYPCHQHASAFDQAARSRGSNRRTALRPRRQQEATEVR LEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYT TQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDR HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPG ERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIG DTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGRDALLQLTSG MVQTHVTTPGSIPTICDLARTFAREMGEAN SEQ ID NO: 5 - Escherichia coli Desaminase (EC-CD)

[0272] MSNNALQTIINARLPGEGLWQIHLQDGKISAIDAQSGVMP ITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVK QRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSY PNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQS RFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQG RFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQ INDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIAS TQPAQTTVYLEQPEAIDYKR SEQ ID NO: 6 - Caspase 9 humana truncada

[0273] GFGDVGALESLRGNADLAYILSMEPCGHCLINNNVNFCRES GLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILS Petition: 870240080912, on September 23, 2024, page. 111 / 136 105 / 105 HGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFE VASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPK SGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

[0274] All patent publications and documents disclosed or mentioned herein are incorporated by reference in their entirety. The foregoing description is provided for illustrative and descriptive purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims. Petition 870240080912, dated 09 / 23 / 2024, pp. 112 / 136

Claims

1 / 2 CLAIMS 1. METHOD FOR GENERATING A HYPOIMMUNOGENIC PLURIPOTENT STEM CELL, characterized by comprising: a. eliminating the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); b. eliminating the activity of both alleles of a CIITA gene in said iPSC; and c. increasing the cell surface expression of CD47 in said iPSC by at least 3.5 times, wherein the increased expression of CD47 reduces the susceptibility of said pluripotent cell to destruction by natural killer (NK) cells.

2. METHOD, according to claim 1, characterized by the increased expression of CD47 on the cell surface being sufficient to allow the hypoimmunogenic pluripotent cell or a differentiated hypoimmunogenic pluripotent cell to survive for at least 2 weeks after transplantation in an individual.

3. METHOD, according to either claim 1 or 2, characterized by increased cell surface expression of the CD47 protein resulting from modification at an endogenous CD47 gene locus or from the expression of a CD47 transgene.

4. METHOD, according to any one of claims 1 to 3, characterized by increased cell surface expression of a CD47 protein resulting from the exchange of an endogenous promoter for a constitutive promoter or an inducible promoter.

5. METHOD, according to any one of claims 1 to 4, characterized in that the HLA-I function is reduced by reducing the expression of the β-2 microglobulin protein or by eliminating a gene encoding a β-2 microglobulin protein.

6. METHOD, according to any one of claims 1 to 5, characterized in that HLA-II function is reduced by reducing the expression of the CIITA protein or by eliminating a gene encoding a CIITA protein.

7. METHOD, according to any one of claims 1 to 6, characterized by comprising: (a) eliminating the HLA-I function in the pluripotent cell; (b) eliminating the HLA-II function in the pluripotent cell; or (c) eliminating both the HLA-I and HLA-II functions.

8. METHOD, according to any one of claims 1 to 7, characterized by further comprising the introduction of a safety switch.

9. METHOD, according to claim 8, characterized in that the safety switch is a suicide gene that is activated by a trigger that causes the hypoimmunogenic pluripotent cell to die.

10. METHOD, according to claim 9, characterized by: (a) the suicide gene being a thymidine kinase gene of herpes simplex virus (HSV-tk) and the trigger being ganciclovir; (b) the suicide gene being a cytosine deaminase gene of Escherichia coli (EC-CD) and the trigger being 5-fluorocytosine (5-FC); (c) the suicide gene encoding an inducible Caspase protein and the trigger being a chemical dimerization inducer (CID). Petition 870260021971, dated 10 / 03 / 2026, p. 12 / 25

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