Immunoengineered pluripotent cells

Abstract

To provide a method of generating a hypo-immunogenic pluripotent stem cell.SOLUTION: Provided is a method of generating a hypo-immunogenic pluripotent stem cell, comprising: eliminating activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); eliminating activity of both alleles of a CIITA gene in the iPSC; and increasing expression of CD47 in the iPSC. In one aspect, the iPSC is human, the B2M gene is human, the CIITA gene is human, and the increased CD47 expression results from introducing at least one copy of a human CD47 gene under control of a promoter into the iPSC cell.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture of U.S. Provisional Patent Application No. 2013 / 0136644 filed on January 13, 2017. The benefit of Application No. 62 / 445,969 is claimed. [Technical field]

[0002] II. FIELD OF THE PRESENTINVENTION Regenerative cell therapy is a key method for regenerating damaged organs and tissues. The scarcity of organs available for transplantation and the resulting Depending on the type of cell line, there may be a long wait time, making it difficult to transfer a readily available cell line to a patient. The ability to regenerate tissue by transplanting regenerative cells is clearly attractive. The therapy has been shown to repair damaged tissue after transplantation in animal models (e.g., after myocardial infarction). However, there are concerns that the immune system of the transplant recipient may be at increased risk of developing pulmonary embolism. The tendency to reject allogeneic substances greatly reduces the inherent efficacy of therapeutic agents As a result, the potentially positive effects surrounding such treatment are diminished. [Background technology]

[0003] III. BACKGROUND OF THE PRESENTINESS Regenerative cell therapy is a method for the regeneration of damaged organs and tissues. This is an important and promising treatment option. The scarcity of organs available for transplantation and the This can result in long wait times and the need to provide readily available cell lines to patients. The ability to regenerate tissue by transplantation is clearly attractive. Cell therapy has been shown to repair damaged tissue after transplantation in animal models (e.g., after myocardial infarction). However, the immune system of transplant recipients has shown promising early results. The tendency of the immune system to reject allogeneic materials greatly reduces the inherent efficacy of therapeutic agents. This reduces the likelihood of side effects occurring, and potentially positive outcomes surrounding such treatment.

[0004] Autologous induced pluripotent stem cells (iPSCs) provide an unrestricted source of patient-specific cell-based organ repair strategies However, producing them is a technical challenge. The lengthy process is complicated by complex manufacturing challenges that conceptually preclude any acute care treatment. Allogeneic iPSC-based therapies are easier from a manufacturing standpoint and are well-suited for transplantation. This allows for the production of cleaned, standardized, high-quality cell products. However, because they are of allogeneic origin, such cell products are unlikely to cause rejection. If we could reduce or eliminate the antigenicity of cells, Pluripotent stem cells can be used to generate pluripotent cell products that are compatible with the three germ layers. Because stem cells can differentiate into any cell type in the body, the potential applications of stem cell therapy are numerous. Differentiation, ex vivo Progenitor cells that continue to differentiate and mature in the organ environment at the transplant site or in the By transplanting cells In vivo And it can be implemented. ex vivo According to differentiation, This allows the researcher or clinician to closely monitor the procedure and ensure proper This allows for the assurance that the appropriate cell population is being produced.

[0005] However, in most cases, undifferentiated pluripotent stem cells are not differentiated because they are prone to form teratomas. These therapies have been avoided in clinical transplantation therapy due to their tendency to cells (e.g., stem cell-derived cardiomyocytes that are transplanted into the myocardium of patients suffering from heart failure) If such pluripotent cells or tissues are applied to clinical practice, The study would benefit from a “safety profile” that would control cell proliferation and survival after transplantation. Wax.

[0006] The art is replete with techniques for producing cells that can be used to regenerate or replace diseased or defective cells. Pluripotent stem cells (PSCs) proliferate rapidly and can produce many types of cells. PSCs can differentiate into various types of cells, so they may be useful for Lee has several antibodies produced by different techniques and with different immunogenic characteristics. The compatibility of patients with engineered cells or tissues derived from PSCs may be important. This determines the risk of immune rejection and the need for immunosuppression.

[0007] Embryonic stem cells (ESCs) isolated from the inner cell mass of blastocysts mismatch with recipients. This immunological barrier is characterized by the presence of histocompatibility antigens, which are a type of antigen that is a match for the human leukocyte antigen (HLA) type. This cannot be solved by ESC banks because non-HLA molecules function as minor antigens. Because of the mismatch, even HLA-matched PSC grafts can be rejected. So far, PSC-based approaches have been successful in preclinical trials, but this is due to In immunosuppressed or immunodeficient models, or by encapsulating cells This has only been achieved by protecting the host's immune system. The systemic immunosuppression used in organ transplantation is justified for the purpose of regenerative approaches. Immunosuppressants have serious side effects and significantly increase the risk of infection and malignancies. Increase.

[0008] To circumvent the rejection problem, various techniques have been developed to generate patient-specific pluripotent stem cells. These include the transfer of a somatic cell nucleus into an enucleated oocyte (somatic cell nuclear transfer ( SCNT (stem cell neoplasia), fusion of somatic cells and ESCs (hybrid cells), and the expression of specific transcription factors This includes reprogramming of somatic cells using artificial PSCs or iPSCs. Although SCNT stem cells and iPSCs are chromosomally identical, they have the same nuclear or cell dots, respectively. SCNT stem cells are derived from mitochondria inherited from oocytes. mtDNA-encoded proteins are involved in the It may act as an antigen and cause rejection. Culture expansion-associated DNA and mtDNA mutations and genetic instability may also lead to immune rejection. It is possible that minor antigens associated with immune responses may be produced. Therefore, it is expected that SCNT stem cells or iPSCs can be used to successfully generate matched patient-specific tissues on a large scale. This reduces the possibility of modifying the model.

[0009] IV. SUMMARY OF THE PRESENT DISCLOSURE A method for treating hypoimmune diseases by avoiding rejection by the host's allogeneic immune system Hypoimmune pluripotent (HIP) cells were generated from maternal blood and fetal tissue. We used placental syncytiotrophoblast cells, which form the interface between MHC I or HLA-I and Decreased expression of MHC II or HLA-II. Increased CD47. Impaired antigen-presenting ability. This pattern of immune response and protection from innate immune clearance may This is due to the fact that HIP cells, as well as the specific differentiation characteristics of HIP cells, avoided the immune rejection reaction of the host. It has been shown to occur in cells of defined ectodermal, mesodermal, and endodermal origin.

[0010] Accordingly, the present invention provides a method for producing hypoimmunogenic pluripotent stem cells, comprising: abolishing the activity of both alleles of the B2M gene in iPSCs; abolishing the activity of both alleles of the CIITA gene in the iPSCs; and 47 ;

[0011] In a preferred embodiment of the method, the iPSCs are human and the B2M gene is human. wherein the CIITA gene is human, and the increased expression of CD47 is At least one copy of the human CD47 gene under the control of a transcription factor is introduced into the iPSC cells. In another preferred embodiment of the method, the iPSCs are mouse. the B2m gene is mouse, the Ciita gene is mouse, and the increased Expression of Cd47 was enhanced by at least one of the promoters of the mouse Cd47 gene. In a more preferred embodiment, the method results from the introduction of at least one copy of the gene encoding the iPSC cell. The promoter is a constitutive promoter.

[0012] In some embodiments of the methods disclosed herein, both alleles of the B2M gene are A cluster in which the disruption in a gene disrupts both alleles of the B2M gene. Clustered Regularly Interspaced Short Palindromic Repeats This method is also used in other In embodiments, the disruption in both alleles of the CIITA gene is This occurs in a CRISPR reaction that destroys both alleles of a gene.

[0013] The present invention provides human hypoimmunogenic pluripotent (hHIP) stem cells that contain: one or more alterations that inactivate one or more alleles of the endogenous CIITA gene; and increasing expression of the CD47 gene in said hHIP stem cells. wherein said hHIP stem cells contain said B2M and CIITA modifications but said C The second induced by induced pluripotent stem cells (iPSCs) does not contain increased expression of the D47 gene Induce a primary natural killer (NK) cell response that is lower than the primary NK cell response of wherein the first and second NK cell responses are induced by the hHIP cells or the B2M and CI cells. Any of the iPSC cells containing ITA modifications but not increased expression of the CD47 gene. both In vitro By determining the IFN-γ levels from NK cells incubated with Provides a measure of

[0014] The present invention provides human hypoimmunogenic pluripotent (hHIP) stem cells, which contain: one or more alterations that inactivate one allele of the endogenous CIITA gene; one or more modifications that inactivate the CD47 gene in said hHIP stem cells; and wherein said hHIP stem cells are modified to include allogeneic humanized iPSC-induced The first T cell response in the humanized mouse strain is lower than the second T cell response in the humanized mouse strain. and wherein said first and second T cell responses are inducible by an immune response to an elimination of the immune response. IFN-γ levels from splenocytes of the humanized mice were determined by Elispot assay. is measured by providing.

[0015] The present invention relates to the use of the hHIP stem cells or derivatives thereof disclosed herein in human subjects. The present invention further provides a method for treating a disease requiring cell transplantation. Use of the hHIP stem cells disclosed herein for the preparation of a medicament for the treatment of provide.

[0016] The present invention relates to low immunogenic pluripotent cells, including: Endogenous major histocompatibility antigen class I (HLA-I) function; reduced when compared to parental pluripotent cells endogenous major histocompatibility complex class II (HLA-II) function; and resistance to NK cell-mediated killing. wherein said hypoimmunogenic pluripotent cells have increased CD47 function, which reduces susceptibility to H Decreased HLA-I function, decreased HLA-II function, and reduced susceptibility to killing by NK cells. As a result of the reduction, the cells are less susceptible to rejection when transplanted into a subject.

[0017] In some embodiments, the hypoimmunogenic pluripotent cells are β-2 microglobulin protein. In a preferred embodiment, the expression of the β-2 miR protein is decreased. The gene that codes for the cycloglobulin protein was knocked out. In a more preferred embodiment, the beta-2 microglobulin protein has the sequence In a more preferred embodiment, the sequence of the first amino acid sequence is at least 90% identical to that of the first amino acid sequence. The beta-2 microglobulin protein has the sequence set forth in SEQ ID NO:1.

[0018] In some embodiments, the HLA-I function comprises reducing expression of HLA-A protein. In a preferred embodiment, the gene encoding the HLA-A protein is In some embodiments, the HLA-I function is knocked out. In a preferred embodiment, the expression of HLA-B protein is decreased. In some embodiments, the HLA protein is knocked out. The HLA-I function is reduced by decreasing the expression of the HLA-C protein. In this form, the gene encoding the HLA-C protein has been knocked out.

[0019] In another embodiment, the hypoimmunogenic pluripotent cells do not contain HLA-I function.

[0020] The present invention relates to a method for reducing the HLA-II function by reducing the expression of CIITA protein. In a preferred embodiment, the present invention provides a method for producing a pluripotent cell having low immunogenicity, comprising the steps of: In a more preferred embodiment, the gene encoding the CII The TA protein has at least 90% sequence identity to SEQ ID NO:2. In a preferred embodiment, the CIITA protein has the sequence of SEQ ID NO:2.

[0021] In some embodiments, the HLA-II function reduces expression of HLA-DP proteins. In a preferred embodiment, the gene encoding the HLA-DP protein is In some embodiments, the HLA-II function is mediated by HLA-DR In a preferred embodiment, the expression of the HL protein is decreased. The gene encoding the A-DR protein is knocked out. reduces the HLA-II function by decreasing the expression of HLA-DQ protein In a preferred embodiment, the gene encoding the HLA-DQ protein is knocked out. It is being done.

[0022] The present invention provides low immunogenic pluripotent cells that do not contain HLA-II function.

[0023] The present invention relates to a method for the treatment of cancer, comprising the steps of: The present invention provides a method for the production of pluripotent cells having low immunogenicity, comprising the steps of: In some embodiments, the increase in expression of the CD47 protein is In another embodiment, the increase results from an alteration to the endogenous CD47 locus. In a preferred embodiment, the increased expression of the protein results from a CD47 transgene. The protein has at least 90% sequence identity to SEQ ID NO:3. In another embodiment, the CD47 protein has the sequence of SEQ ID NO:3.

[0024] The present invention provides a method for the production of pluripotent cells that are activated by a trigger to kill the pluripotent cells or their differentiated progeny. In a preferred embodiment, the present invention provides a hypoimmunogenic pluripotent cell comprising a suicide gene that is activated. the suicide gene is herpes simplex virus thymidine kinase gene (HSV-tk); In a more preferred embodiment, the trigger is ganciclovir. encodes a protein that contains at least 90% sequence identity to SEQ ID NO:4. In a more preferred embodiment, the HSV-tk gene is a protein comprising the sequence of SEQ ID NO:4. Code the following:

[0025] In another preferred embodiment, the suicide gene is the Escherichia coli cytosine deaminase gene (EC -CD) and the trigger is 5-fluorocytosine (5-FC). The EC-CD gene is a gene that has at least 90% sequence identity to SEQ ID NO:5. In a more preferred embodiment, the EC-CD gene encodes a protein represented by SEQ ID NO:5. It encodes a protein comprising the sequence:

[0026] In another preferred embodiment, the suicide gene encodes an inducible caspase protein. In a more preferred embodiment, the trigger is a chemical inducer of dimerization (CID). The inducible gene is an inducible caspase having at least 90% sequence identity to SEQ ID NO:6. In a more preferred embodiment, the gene encodes a ribozyme protein. In a more preferred embodiment, the inducible caspase protein comprises the sequence The CID is AP1903.

[0027] The present invention provides a method for reducing endogenous major histocompatibility antigen class I (HLA-I) function in pluripotent cells. Reduces endogenous major histocompatibility antigen class II (HLA-II) function in pluripotent cells and inhibiting said multipotency against phagocytosis by macrophages or killing by NK cells. Increase expression of proteins that decrease the susceptibility of pluripotent cells; including low immunogenicity A method for producing cells is provided.

[0028] In one embodiment of this method, the HLA-I function is expression of the β-2 microglobulin protein. In a preferred embodiment, the β-2 microglobulin is Expression of the phosphoprotein is associated with the expression of the gene encoding the β-2 microglobulin protein. In a more preferred embodiment, the β-2 miR gene is knocked out. The IgG1 protein has at least 90% sequence identity with SEQ ID NO:1. In a more preferred embodiment, the beta-2 microglobulin protein has the sequence of SEQ ID NO:1. It has an array.

[0029] In another embodiment of this method, the HLA-I function is reduced expression of HLA-A protein expression. In a preferred embodiment, the HLA-A protein expression is decreased by The effect of knocking out the gene encoding the HLA-A protein is to reduce the In another embodiment of the method, the HLA-I function reduces expression of HLA-B protein expression. In a preferred embodiment, the HLA-B protein expression is reduced by The effect of this method is to knock out the gene encoding the HLA-B protein. In another embodiment, the HLA-I function is a decrease in expression of HLA-C protein expression. In a preferred embodiment, the HLA-C protein expression is reduced by It is reduced by knocking out the gene that codes for the protein.

[0030] In another embodiment of the method, the hypoimmunogenic pluripotent cells do not contain HLA-I function.

[0031] In another embodiment of the method, the HLA-II function reduces expression of CIITA protein. In a preferred embodiment, the expression of the CIITA protein is reduced by It is reduced by knocking out the gene encoding the CIITA protein. In a preferred embodiment, the CIITA protein has a sequence identity of at least 90% to SEQ ID NO:2. In a more preferred embodiment, the CIITA protein has sequence identity to SEQ ID NO: It has the sequence of 2.

[0032] In another embodiment of this method, the HLA-II function reduces expression of HLA-DP protein. In a preferred embodiment, the expression of the HLA-DP protein is reduced by The effect of knocking out the gene encoding the HLA-DP protein is to reduce the In another embodiment of the method, the HLA-II function comprises decreasing expression of HLA-DR protein. In a preferred embodiment, the expression of the HLA-DR protein is reduced by The method reduces the expression of LA-DR by knocking out the gene encoding the protein. In some embodiments, the HLA-II function reduces expression of HLA-DQ protein. In a preferred embodiment, the expression of the HLA-DQ protein is reduced by It is reduced by knocking out the gene that codes for the HLA-DQ protein.

[0033] In another embodiment of this method, the hypoimmunogenic pluripotent cells do not contain HLA-II function.

[0034] In another embodiment of the method, the pluripotent cells are sensitized to phagocytosis by macrophages. The increased expression of a protein that reduces susceptibility results from an alteration to the endogenous locus. In a preferred embodiment, the endogenous locus encodes the CD47 protein. In an embodiment, the increase in protein expression occurs due to expression of a transgene. In a more preferred embodiment, the transgene encodes a CD47 protein. wherein the CD47 protein has at least 90% sequence identity to SEQ ID NO:3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO:3.

[0035] Another embodiment of the method comprises activating a trigger that kills the poorly immunogenic pluripotent cells. In a preferred embodiment, the method further comprises expressing a suicide gene that is is the herpes simplex virus thymidine kinase gene (HSV-tk), and the trigger is In a more preferred embodiment, the HSV-tk gene is SEQ ID NO:4. The present invention relates to a method for producing a protein having at least 90% sequence identity to a target gene. In an embodiment, the HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:4.

[0036] In another embodiment of this method, the suicide gene is the Escherichia coli cytosine deaminase gene (EC -CD) and the trigger is 5-fluorocytosine (5-FC). In a preferred embodiment The EC-CD gene is a protein having at least 90% sequence identity to SEQ ID NO:5. In a more preferred embodiment, the EC-CD gene encodes a protein having the sequence of SEQ ID NO:5. The protein encodes a protein comprising the sequence.

[0037] In another embodiment of this method, the suicide gene encodes an inducible caspase protein. wherein the trigger is a specific chemical inducer of dimerization (CID). In one embodiment, the gene is an inducible gene comprising at least 90% sequence identity to SEQ ID NO:6. In a more preferred embodiment, the gene encodes a caspase protein. A more preferred embodiment of the present invention is an inducible caspase protein comprising the sequence of In the example, the CID is AP1903. [Brief description of the drawings]

[0038] V. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]FIG. 1A shows the rationale for the novel hypoimmune pluripotent cells described herein. The fetus is protected from "rejection" during pregnancy by feto-maternal tolerance. The cells have downregulated expression of MHC class I. The cells also have downregulated expression of MHC class II. The cells also have upregulated CD47. FIG. 1B shows that feto-maternal tolerance is mediated by syncytiotrophoblast cells. FIG. 1C shows that syncytiotrophoblast cells have no expression of MHC I and II and high CD47 expression levels. [Diagram 2] Figure 2 shows mouse induced pluripotent stem cells (miPSCs) generated from C57BL / 6 fibroblasts. Pluripotency was verified by reverse transcriptase polymerase chain reaction (rtPCR). Multiple mRNAs associated with pluripotency were detected in miPSC cell extracts but not in uninduced cells (parental mouse fibroblasts). [Diagram 3] Figure 3 confirms the pluripotency of miPSC cells. The C57BL / 6 miPSC cells formed teratomas in syngeneic mice as well as in BALB / c nude and scid beige mice. No teratomas were formed in immunocompetent allogeneic BALB / c mice. [Figure 4] Figure 4 shows that when β-2-microglobulin expression was knocked out in miPSC cells, MHC-I expression could not be induced by stimulation with IFN-γ (right panel). As a control, stimulation of parental miPSC cells with IFN-γ (left panel) increased their MHC-I expression. [Diagram 5] FIG. 5 shows that miPSC / β-2-microglobulin knockouts that also include knockout of Ciita expression (double-knockout) show no baseline MHC-II expression and cannot induce MHC-II expression by TNF-α. [Figure 6] Figure 6A shows increased expression of Cd47 from the transgene in addition to the β-2-microglobulin / Ciita double knockout (iPSChypo cells), and Figure 6B shows that C57BL / 6 iPSChypo cells survive in an allogeneic BALB / c environment, whereas parental iPSC cells do not. [Figure 7] Figure 7 shows one embodiment of the present invention. It shows a schematic diagram of the modification of iPSCs to produce hypoimmune pluripotent cells of the present invention. To generate hypoimmune stem cells, first, both B2m alleles were knocked out using CRISPR-Cas 9 modification. Next, both Ciita gene alleles were knocked out using CRISPR-Cas 9 modification. As a third step, the Cd47 gene was knocked in using lentivirus. [Figure 8] Figure 8A shows a schematic of the role of B2m in the MHC I complex. Knocking out B2m results in loss of MHC I in mice or HLA-1 in humans. Figure 8B shows a schematic of Ciita being a transcription factor that drives expression of MHC II in mice or HLA-II in humans. Knocking out Ciita results in loss of expression of MHC II or HLA-II. [Figure 9] Figures 9A, 9B and 9C show that B2m- / - iPSCs lack expression of MHC-1, B2m- / - Ciita- / - iPSCs lack expression of MHC-1 and MHC-II, and B2m- / - Ciita- / - Cd47 tg iPSCs lack expression of MHC-I and MHC-II and overexpress Cd47. [Figure 10]Figures 10A, 10B, 10C, 10D and 10E show mouse models of "wild type iPSCs" versus immune-lowered PSCs transplanted into allogeneic or syngeneic host mice, where the iPSCs were generated from C57BL / 6 mice and the allogeneic mice were BALB / c. Figure 10A, "wild type iPSCs" formed teratomas only in the femur of syngeneic C57BL / 6 mice. In contrast, there was an immune response in the allogeneic host mice (BALB / c) and no teratomas developed. Figure 10B, "wild type iPSCs" formed teratomas in syngeneic C57BL / 6 mice. Figure 10C, the immune response inhibited teratomas formation in allogeneic BALB / c mice. Figure 10D compares T cell responses (IFN-γ and IL-4) to iPSCs in syngeneic and allogeneic hosts using a spot frequency assay (frequency of cells releasing IFN-γ and IL-4). Release of IFN-γ and IL-4 was very low in C57BL / 6 hosts but dramatically increased in BALB / c hosts. Figure 10E shows B cell responses in syngeneic and allogeneic hosts. The iPSCs were incubated with serum from host animals that had previously received iPSCs transplants. Bound immunoglobulins were measured using flow cytometry. Mean fluorescence intensity (MFI) was significantly higher in serum from allogeneic BALB / c recipient hosts. [Figure 11]Figures 11A, 11B, 11C, 11D and 11E show that knocking out the B2m gene in the iPSCs was partially effective. Figure 11A, B2m- / - iPSCs grew in the thighs of syngeneic C57BL / 6 mice and formed teratomas due to lack of immune response, whereas there was a partial immune response in allogeneic host mice (BALB / c); for example, some of the transplanted cells survived. Figure 11B, B2m- / - iPSCs formed teratomas in syngeneic mice. Figure 11C, partial survival (60%) was observed in the allogeneic host. Figure 11D, differences in T cell responses (IFN-γ and IL-4) between the two hosts indicate a small but detectable T cell response to B2m- / - iPSCs. Figure 11E shows the B cell responses in different host mice, indicating a weaker immune response than against wild-type iPSCs. Immunoglobulin responses were still significantly stronger after allogeneic transplantation of B2m- / - iPSCs into BALB / c compared to syngeneic transplantation into C57BL / 6. Thus, B2m- / - iPSCs had limited survival in allogeneic recipients. [Figure 12]Figures 12A, 12B, 12C, 12D and 12E show that knocking out the B2m and Ciita genes in iPSCs partially increases the effect of cell survival in syngeneic and allogeneic host mice. Figure 12A, B2m- / - Ciita- / - iPSCs formed teratomas in the thighs of syngeneic C57BL / 6 mice due to the absence of immune response, whereas in allogeneic host mice (BALB / c), there was a partial immune response (but reduced compared to B2m- / - iPSCs). Figure 12B, B2m- / - Ciita- / - iPSCs formed teratomas in syngeneic mice. Figure 12C shows that some cell grafts (91.7%) survive in the allogeneic host. Figure 12D shows the difference in T cell responses (IFN-γ and IL-4) between the two hosts, with a slightly higher IFN-γ response in allogeneic recipients compared to syngeneic recipients. Figure 12E shows the B cell response in the different host mice. Compared to wt iPSCs and B2m- / - iPSCs, there was a weaker immune response. No significant differences were observed between allogeneic and syngeneic recipients. Overall, B2m- / - Ciita- / - iPSCs had limited survival in allogeneic recipients, which could be attributed to a measurable immune response. [Figure 13]Figures 13A, 13B, 13C, 13D and 13E show the complete effect of knocking out B2m and Ciita genes in iPSCs and knocking in the Cd47 transgene on cell survival in syngeneic and allogeneic host mice. Figure 13A, B2m- / - Ciita- / - Cd47tg iPSCs teratomas grew in both syngeneic C57BL / 6 and allogeneic host thighs. 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 responses (IFN-γ and IL-4) in allogeneic recipients. No differences were observed between the two hosts. Figure 13E shows the lack of B cell responses in allogeneic recipients. No differences were observed between the two hosts. Thus, survival of B2m- / - Ciita- / - Cd47tg iPSCs was complete in allogeneic recipients. They were not immunogenic as they did not induce T or B cell responses. [Figure 14]Figures 14A, 14B, and 14C show that B2m- / - Ciita- / - Cd47tg iPSCs (referred to as hypoimmunogenic pluripotent cells (HIP) cells) evaded the host immune system. Figure 14A, expression of stimulatory NK cell ligands was not increased in HIP cells. Fusion proteins recognizing various ligands of the NK cell transmembrane protein NKG2D were used to assess the levels of activating ligands that can activate cytolytic NK cell activity. Thus, binding of fusion proteins to iPSCs is a global parameter for the expression of ligands that activate NKG2D. Figure 14B, HIP cells did not induce NK cells to increase expression of CD107a (a marker of functional NK cell activity). In contrast, B2m- / - Ciita- / - iPSCs induced the expression of CD107a on NK cells, thereby triggering the cytolytic function of NK cells. Figure 14C. IFN-γ ELISpot assays using purified syngeneic NK cells from C57BL / 6 mouse spleens showed no NK cell response induced by HIP cells. Thus, NK cells were not activated to release IFN-γ. The spot frequency of HIP cells was not different from that of unstimulated NK cells (negative control). Only B2m- / - Ciita- / - iPSCs significantly increased the IFN-γ spot frequency. [Figure 15]Figures 15A and 15B show further data showing that the HIP cells avoided rejection or killing by the innate immune system due to the Cd47 transgene. In an in vivo NK cell assay, a mixture of 50% iPSCs and 50% HIPs was injected into the NK-rich peritoneum of syngeneic C57BL / 6 mice, where cytotoxicity is caused by NK cells. After 24 and 48 hours, peritoneal cells were harvested and sorted. Figure 15A compares the iPSCs with B2m- / - Ciita- / - iPSCs (without Cd47 transgene). The B2m- / - Ciita- / - iPSCs were selectively killed by NK cells. Figure 15B compares iPSCs with B2m- / - Ciita- / - Cd47 tg iPSCs (HIP cells). The HIP cells were not selectively killed by NK cells. The proportion of HIP cells among peritoneal iPSCs remained at 50%, indicating the absence of NK cell stimulation. Thus, knockout of MHC-I and MHC-II rendered the cells highly susceptible to killing by NK cells, whereas overexpression of the Cd47 abrogated the interaction with stimulatory NK cells. [Figure 16] FIG. 16 shows that the mouse HIP cells of the present invention exhibited a normal mouse karyotype. [Figure 17]Figure 17A, Figure 17B and Figure 17C show that the mouse HIP cells of the present invention maintained pluripotency during the modification process. RT-PCR analysis of markers commonly accepted to indicate pluripotency (Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1, and actin as a loading control) is shown. The pluripotency markers were expressed throughout the three-step modification process. Figure 17A compares iPSCs, B2m- / - iPSCs, and mouse fibroblasts (negative control). B2m- / - iPSCs maintained pluripotency genes. Figure 17B shows the same analysis but with B2m- / - Ciita- / - iPSCs. They maintained the same pluripotency genes. Figure 17C shows the same analysis but with B2m- / - Ciita- / - Cd47 tg iPSCs (HIP cells). These cells carried the same pluripotency genes. Furthermore, histology of teratomas that developed after transplantation of HIP cells into SCID-beige mice indicates that cell types associated with ectoderm, mesoderm, and endoderm were identified. Immunofluorescent markers for all three germ layers were detected (data not shown). Cell morphology was correct for neuroectoderm, mesoderm, and endoderm. Immunofluorescent staining for DAPI, GFAP, cytokeratin 8, and brachyury confirmed the pluripotency of HIP cells. [Figure 18]Figures 18A, 18B, and 18C show that HIP cells differentiated into mesodermal lineage cells and lost pluripotency markers of HIP cells. Figure 18A shows that pluripotency markers in HIP cells (labeled "mHIP") are lost in differentiated mouse endothelial cells (labeled "miEC"). Figure 18B shows that the pluripotency markers are retained in HIP cells but not in differentiated mouse smooth muscle cells (labeled "miSMC"). Figure 18C shows that the pluripotency markers are retained in HIP cells but not in differentiated mouse cardiomyocyte cells (labeled "miCM"). These results were confirmed by immunohistochemistry (data not shown). Endothelial cells were detected with anti-CD31 and anti-VE-cadherin antibodies, smooth muscle cells were detected with anti-SMA and anti-SM22 antibodies, and cardiomyocytes were detected with anti-troponin I and anti-sarcoma alpha-actinin antibodies. [Figure 19] Figures 19A and 19B show that the HIP cells differentiated into endodermal lineage islet cells (iICs) that produced C-peptide and insulin. Figure 19A, differentiation markers were not detected in HIP cells but were detected in induced islet cells. Figure 19B, induced islet cells produced insulin. Immunohistochemical staining for C-peptide confirmed these results (data not shown). [Figure 20] Figures 20A and 20B show that the HIP cells differentiated into ectodermal lineages. Figure 20A shows HIP cells in vitro, and Figure 20B shows differentiated neural cells. Immunohistochemical staining with the neuroectodermal stem cell markers Nestin and Tuj-1 confirmed these results (data not shown). [Figure 21]Figures 21A, 21B and 21C show that cells differentiated from the HIP cells retained the phenotype of lacking MHC I and II expression and overexpression of Cd47. Figure 21A compares the expression of MHC-I, MHC-II and Cd47 between mouse induced endothelial cells ("miEC") and B2m- / - Ciita- / - Cd47 tg miEC cells. Figure 21B compares the expression of MHC-I, MHC-II and Cd47 between mouse induced smooth muscle cells ("miSMC") and B2m- / - Ciita- / - Cd47 tg miSMC cells. Figure 21C compares the expression of MHC-I, MHC-II and Cd47 between mouse induced cardiomyocytes ("miCM") and B2m- / - Ciita- / - Cd47 tg miCM cells. [Figure 22] Figures 22A, 22B, and 22C show that endothelial cells differentiated from the HIP cells are less immunogenic. Figure 22A, Transplantation of C57BL / 6 miECs or miECs differentiated from mHIP into syngeneic and allogeneic mice. Figure 22B, miECs in allogeneic but not syngeneic BALB / c recipient mice elicited a significant immune response, as evidenced by strong IFN-γ ELISPOT and immunoglobulin responses (FACS analysis) in BALB / c recipients. Figure 22C, miEC cells differentiated from HIP did not elicit an immune response in syngeneic or allogeneic recipients. [Figure 23]Figures 23A, 23B, and 23C show that mouse-derived smooth muscle cells differentiated from HIP cells are less immunogenic. Figure 23A, Transplantation of C57BL / 6 miSMCs or miSMCs differentiated from HIP into syngeneic and allogeneic mice. Figure 23B, miSMCs in allogeneic but not syngeneic mice elicited a significant immune response, as evidenced by strong IFN-γ ELISPOT and immunoglobulin responses (FACS analysis) in BALB / c recipients. Figure 23C, miSMC cells differentiated from HIP did not elicit an immune response in syngeneic or allogeneic recipients. [Figure 24] Figures 24A, B, and C show that mouse-derived cardiomyocytes differentiated from HIP cells are hypoimmunogenic. Figure 24A, Transplantation of C57BL / 6 miCMs or miCMs differentiated from HIP into syngeneic and allogeneic mice. Figure 24B, miCMs in allogeneic but not syngeneic BALB / c recipient mice elicited a significant immune response, as evidenced by strong IFN-γ ELISPOT and immunoglobulin responses (FACS analysis) in BALB / c recipients. Figure 24C, miCMs differentiated from HIP did not elicit an immune response in syngeneic or allogeneic recipients. [Diagram 25] Figure 25 shows that differentiated cells derived from HIP cells (miECS, miSMCs, miCMs) avoided rejection via the innate immune system. NK fusion protein assays showed that none of the three differentiated cells increased expression of stimulatory NK cell ligands when compared to differentiated cells derived from miPSCs. [Figure 26]Figures 26A and 26B show that miECs derived from HIP cells of the present invention evaded immune responses and survived long-term in allogeneic hosts. Figure 26A, miPSCs-derived miEC grafts survived long-term in syngeneic recipients (C57BL / 6) but were rejected in allogeneic recipients (BALB / c). Figure 26B, HIP-derived miECs survive long-term after transplantation in both syngeneic and allogeneic recipients. [Figure 27] Figure 27: HIP cell-derived miECS organized to form vasculature in allogeneic host. After implantation into Matrigel matrix, the miECs organized three-dimensionally to form vasculature over a period of 6 weeks. These results were confirmed by immunofluorescent staining for luciferase and VE-cadherin; prior to implantation, miECs were transduced to express luciferase. Survival was monitored by bioluminescence imaging, and implanted cells were identified by immunofluorescent staining for luciferase (data not shown). [Figure 28] FIG. 28 shows that the human HIP cells exhibited a normal human karyotype. [Figure 29] Figure 29 shows that human HIP cells maintained pluripotency during the modification process. The hiPSCs (e.g., starting cells prior to modification of the present invention) and the HIP cells both have expression of pluripotency genes (NANOG, OCT4, SOX2, DPPA4, hTERT, ZFP42, and DEMT3B; G3PDH as a loading control) using PCR assays. Immunofluorescence staining confirmed this finding, as the cells express TRA-1-60, TRA-1-81, Sox2, Oct4, SSEA-4 markers, and alkaline phosphatase (data not shown). [Diagram 30]Figures 30A and 30B show that 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 production or IL-5 in an ELISPOT assay. In contrast, they did respond to transplanted iPSCs. Figure 30B shows that only iPSCs elicited a strong antibody response by flow cytometry; the HIP cells did not. [Diagram 31] Figures 31A, 31B, 31C, and 31D show that human HIP cells were differentiated into mesodermal lineages. Figure 31A shows the morphology of human HIP cells. Figure 31B shows HIP-derived endothelial cells stained with CD31, VE-cadherin, and DAPI as a control. Figure 31C shows HIP-derived cardiomyocytes stained with α-sarcomeric actinin, troponin I, and DAPI as a control. Figure 31D shows immature stage angiogenesis by the HIP-derived endothelial cells. HIP-derived cardiomyocytes were observed to be beating (data not shown). [Diagram 32] 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 showed significant T cell responses in IFN-γ and IL-5 ELISPOT assays, whereas human HIP cell-derived hiECs did not. Figure 32B shows B cell responses by flow cytometry. Only hiECs produced significant immunoglobulin binding as measured by mean fluorescence intensity (MFI). [Diagram 33]Figures 33A and 33B show that transplantation of human cardiomyocytes derived from human HIP cells did not result in an immune response in allogeneic humanized mice. Figure 33A shows the difference in T cell responses for "wild type" hiCMs and hiCMs derived from B2M- / - CIITA- / - CD47 tg HIP cells in IFN-γ and IL-5 ELISPOT assays. Figure 33B shows the B cell responses in flow cytometry. Only "wild type hiCMs" resulted in significant immunoglobulin loading of hiECs as measured by mean fluorescence intensity (MFI). [Diagram 34] Figures 34A, 34B and 34C show that the derivatives of mouse HIP cells of the present invention evaded the rejection of the innate immune system. NK cells were isolated from BALB / c mice using magnetic activated cell sorting (MACS). 5x106 stimulator cells (C57BL / 6 iPSC B2m- / - Ciita- / - derived cells (derivatives) or iPSC B2m- / - Ciita- / - Cd47 tg derived cells (derivatives)) of either iEC, iSMC or iCM were incubated with 5x106 MACS-sorted NK cells in IFN-γ ELISPOT plates. After 24 hours, the spot frequency was measured by ELISPOT reader. All three cell types derived from B2m- / - Ciita- / - induced strong NK responses. However, all three cell types derived from B2m- / - Ciita- / - Cd47 tg did not induce NK cell responses and their spot frequencies were not statistically different from the negative control (isolated NK cells not incubated with stimulator cells). Figure 34A shows endothelial cells, Figure 34B shows smooth muscle cells, Figure 34C shows cardiomyocytes, and Figure 34D shows the positive control of YAC-1 mouse lymphoma. [Diagram 35]Figures 35A, 35B and 35C show the innate immune response (or lack thereof). A mixture of 50% wild type derived derivatives (5x106 cells) and 50% C57BL / 6 B2m- / - Ciita- / - or B2m- / - Ciita- / - Cd47 tg derived derivatives (5x106 cells) was prepared. The cells were stained with 10 μM CFSE stain for 10 minutes and resuspended in 500 μl saline. 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 hours, peritoneal cells were harvested to sort the cells and their ratios were calculated. Wild type and modified cells were identified in FACS by MHCI staining. Figure 35A shows endothelial cells. Figure 35B shows smooth muscle cells. Figure 35C shows cardiomyocytes. Only B2m- / - Ciita- / - derived derivatives were killed by NK cells, and their ratio was significantly decreased. None of B2m- / - Ciita- / - Cd47 tg derived derivatives were killed by NK cells, and their ratio remained stable at about 50%. [Diagram 36] Figures 36A, 36B and 36C show genetic modification of human iPSCs verified by FACS. The lack of HLA I and HLA II was confirmed in B2M- / - CIITA- / - hiPSCs. Furthermore, B2M- / - CIITA- / - CD47 tg showed high CD47 expression. Figure 36A shows the results for HLA I. Figure 36B shows the results for HLA II. Figure 36C shows the results for CD47. [Figure 37]Figures 37A and B show that the immunophenotype was maintained following differentiation of B2M- / - CIITA- / - CD47 tg iPSCs. FACS analysis showed that B2M- / - CIITA- / - CD47 tg derived derivatives lacked expression of HLA I and HLA II and overexpressed CD47 when compared to unmodified wt derivatives. Figure 37A shows endothelial cells and Figure 37B shows cardiomyocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] VI. DETAILED DESCRIPTION OF THE PRESENTING A. INTRODUCTION The present invention, as outlined herein, In addition, several genetically engineered mice can be engineered to have hypoimmunogenic pluripotency (HypoImmuno) to evade the host immune response. The present invention provides human genic pluripotent (HIP) cells, which are the primary regulators of immune responses. They lack immune antigens and are modified to avoid phagocytosis. This allows them to target specific Derivation of "off-the-shelf" cell products for the generation of tissues and organs This will enable the use of derivatives of human allogeneic HIP cells in human patients. The advantage of being able to use the system is that it can be used in a variety of ways, such as to reduce the long-term complications typically seen in allogeneic transplants. Significant advantages include the avoidance of immunosuppressive therapy and drug use. It also allows the use of cell therapy without the need for individual treatments for each patient. Recently, cells produced from autologous cell sources have A product may be susceptible to immune rejection if it has only a few or one single antigenic variation. Therefore, autologous cell products are not inherently non-immunogenic. In addition, modifying cells and quality control are very time-consuming and expensive, and autologous cells are difficult to use during the acute phase. It is not available as a treatment option. If the immune disorder can be overcome, Only allogeneic cell products could be used in larger patient populations. provides a universal cell supply for generating universally acceptable derivatives. It will serve as a source.

[0040] The present invention relates to taking advantage of the feto-maternal tolerance that exists in pregnant women. Half of the human leukocyte antigens (HLA) in the fetus are paternally inherited, and the fetus has a major HLA mismatch. Although the fetus expresses allogeneic antigens, the maternal immune system does not recognize the fetus as an allogeneic entity and Do not initiate an immune response (such as that seen in "host-versus-graft" type immune responses). Feto-maternal tolerance is mediated primarily by syncytiotrophoblast cells at the feto-maternal interface. As shown in FIG. 7, syncytiotrophoblast cells express major histocompatibility complex I and II (MHC-I and MHC-II) proteins, and CD47 (which “Don’t eat me” suppresses phagocytic innate immune surveillance and clearance of HLA-deficient cells 't eat me) The expression of α-amyloides (ALA) is also increased during pregnancy. The HIP cells of the present invention are produced by the same tolerance mechanism that prevents rejection of the infant. The cells avoid rejection and have long-term survival after allogeneic transplantation, and It also becomes possible for the cells to be engrafted.

[0041] These results demonstrate that this feto-maternal tolerance (compared to starting iPSCs, e.g. hiPSCs) Only three genetic modifications, two reduced activities ("knockouts" as further described herein), were made. One type of increased activity (described herein as a "knock-in") can be introduced into the target cell by a single gene. In general, those skilled in the art have attempted to suppress the immunogenicity of iPSCs. However, there have been only limited successes; Rong et al., Cell Stem Cell 14:121-130 ( 2014) and Gornalusse et al., Nature Biotech doi:10.1038 / nbt.3860 .

[0042] Thus, the present invention provides a method for generating HIP cells from pluripotent stem cells and for maintaining them. , differentiation, and ultimately transfer of those derivatives to patients in need thereof. Provide for planting.

[0043] B. Definitions The term "pluripotent cells" refers to cells that are capable of self-renewal and proliferation while remaining in an undifferentiated state. and cells that can be induced to differentiate into specialized cell types under appropriate conditions. As used herein, the term "pluripotent cells" refers to embryonic stem cells and fetal, amniotic or The term includes other types of stem cells, including somatic stem cells, etc. Exemplary human stem cell lines include H9 human stem cell lines. Further exemplary stem cell lines include the National Institutes of Health's Human Embryonic Stem Cell Line. The National Institutes of Health Human Embryonic Stem Cell Registry The Howard Hughes Medical Institute Registry and the Howard Hughes Medical Institute HUES Collection s Medical Institute HUES collection)(Cowan, CA et. al, New England J. Med. 350:13. (2004), which is incorporated herein by reference in its entirety. This includes those made available through the

[0044] As used herein, "pluripotent stem cells" refer to stem cells that can be derived from any of three germ layers: endoderm (e.g., gastrointestinal (stomach linking), gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital They have the potential to differentiate into either the germ layer (e.g. organ tissue) or the ectoderm (e.g. epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cells" may also be used to refer to "induced pluripotent stem cells," The term "iPSCs" also includes certain pluripotent stem cells derived from non-pluripotent cells. Examples of parent cells include were reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells are capable of expressing specific regulatory genes. Created by induction or by exogenous addition of specific proteins Methods for deriving iPS cells are known in the art and are described further 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., Nature458 (7239): 766-77 0 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009), respectively. (The entire contents of which are incorporated herein by reference.) Generating Induced Pluripotent Stem Cells (iPSCs) As used herein, "hiPSCs" refers to human induced pluripotent stem cells (hiPSCs). and "miPSCs" are mouse induced pluripotent stem cells.

[0045] "Characteristics of pluripotent stem cells" refer to cellular characteristics that distinguish pluripotent stem cells from other cells. Under appropriate conditions, they can commit to cell lineages of all three germ layers (endoderm, mesoderm, and ectoderm). The ability to give rise to progeny that can differentiate into a population of characteristic cell types is a hallmark of pluripotent stem cells. The expression or absence of expression of specific combinations of molecular markers can also be used to characterize pluripotent stem cells. For example, human pluripotent stem cells may be selected from the following non-limiting list: SSEA-3, SSEA-4, SSEA-5, SSEA-6, SSEA-7, SSEA-8, SSEA-9, SSEA-10, SSEA-11, SSEA-12, SSEA-13, SSEA-14, SSEA-15, SSEA-16, SSEA-17, SSEA-18, SSEA-19, SSEA-20, SSEA-21, SSEA-22, SSEA-23, SSEA-24, SSEA-25, SSEA-26, SSEA-27, SSEA-28, SSEA-29, SSEA-30, SSEA-31, SSEA-32, SSEA-33, SSEA-34, SSEA-35, SSEA-36, EA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex 1, and Nanog, and in some embodiments The cellular morphology associated with pluripotent stem cells is also consistent with that of pluripotent stem cells. As described herein, the cells are endoderm progenitor cells and / or hepatocytes. There is no need to go through pluripotency to be reprogrammed into cells.

[0046] As used herein, the term "multipotent" or "multipotent cells" refers to "Artificially differentiated cell" refers to a cell type that can give rise to a limited number of other specific cell types. Induced multipotent cells can form endoderm cells. Multipotent blood stem cells can produce lymphocytes, monocytes, neutrophils, etc. They can differentiate into several types of blood cells, including:

[0047] As used herein, the term "oligopotent" refers to the ability of adult stem cells to The ability to differentiate into several different cell types, e.g. lymphoid or myeloid stem cells. can give rise to cells of either the lymphoid or myeloid lineage, respectively.

[0048] As used herein, the term "unipotent" refers to a cell that is capable of forming a single cell type. For example, spermatogonial stem cells can only form sperm cells.

[0049] As used herein, the term "totipotent" refers to a cell that is capable of forming an entire organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent. It is sexuality.

[0050] As used herein, "non-pluripotent cells" refers to cells that are capable of pluripotency. A mammalian cell that is not a reproductive cell. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, bone marrow, skin, skeletal muscle, adipose Exemplary cell types include cells derived from tissues selected from adipose tissue and peripheral blood. , including but not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, These include osteoclasts, and T cells. Induced multipotent cells, The starting cells used to generate endodermal progenitor cells and hepatocytes are non-pluripotent cells There are times when this happens.

[0051] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, These include, but are not limited to, unipotent cells, progenitor cells, and terminally differentiated cells. In certain embodiments, a less potent cell is compared to a more potent cell: It is considered to be “differentiated.”

[0052] Somatic cells are the cells that make up the body of an organism. Somatic cells include the cells that make up the organs, skin, blood, and bones of an organism. , which contain cells that make up connective tissue, but do not contain germ cells.

[0053] The cells may be derived, for example, from a human or non-human mammal. Mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, and Examples of animals that may be of concern include pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, the cells are derived from an adult human or non-human mammal. In embodiments, the cells are derived from a neonatal human, an adult human or a non-human mammal.

[0054] As used herein, the term "subject" or "patient" refers to any animal, including domestic animals, zoo animals, or The term "subject" or "patient" refers to any animal, including humans. or a mammal, such as a human. Including but not limited to liver, heart, lung, kidney, pancreas, brain, nervous tissue, blood, bone, This includes individuals (particularly humans) who have a disease or disorder related to the bone marrow, etc.

[0055] Mammalian cells may be derived from human or non-human mammals. Exemplary non-human mammals include Mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, and Lumots, hamsters, sheep, pigs, horses, cattle, and non-human primates (e.g., chimpanzees). These include the reptiles, monkeys, macaques, and apes.

[0056] As used herein, the term "low immunogenic pluripotent cells" or "HIP cells" refers to cells that have the pluripotent characteristic. and pluripotent cells that induce reduced immune rejection when transplanted into an allogeneic host. In a preferred embodiment, the HIP cells do not elicit an immune response. "Low immunogenicity" refers to a gene that is less immunogenic than the parent (i.e., "wt") gene prior to immunological modification, as outlined herein. ") The immune response was significantly reduced or absent when compared to the immune response of cells. In many cases, the HIP cells are immunologically silent. Assays for the characteristics of HIP are outlined below. do.

[0057] "HLA" or "human leukocyte antigen" complex refers to the major histocompatibility complex (HLA) that is found in humans. The HLA complex is made up of a complex of genes that code for major histocompatibility complex (MHC) proteins. Cell surface proteins are involved in regulating the immune response to antigens. In humans, class I There are two MHCs, class II and class II, namely, "HLA-I" and "HLA-II". HLA-I binds to the Peptide presentation from the inside and antigens presented by HLA-I complexes induce killer T cells (also known as CD8+ T cells or cytotoxic T cells) The HLA-1 protein is the β-2 microglobulin. HLA-II is a receptor that binds to the extracellular antigen-presenting T lymphocytes. It contains five proteins (HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR). stimulates CD4+ cells (also known as T helper cells). "MHC" or "HLA" Whichever is used, it depends on whether the genes are human (HLA) or mouse (MHC) derived. It should be understood that the above is not meant to be limiting, as it depends on the As it relates to mammalian cells, these terms may be used interchangeably herein.

[0058] As used herein, "gene knockout" refers to the knockout of a particular gene by isolating it from the host cell in which it resides. Inactivate the target protein in the cells so that the protein is not produced or inactivated As will be understood by those of skill in the art and as further described below, This can involve removing nucleic acid sequences from genes, interrupting sequences with other sequences, A number of different methods, including altering the frame or modifying the nucleic acid of a regulatory component, etc. This can be achieved, for example, by removing all or part of the coding region of the gene of interest, or can be replaced with a "nonsense" sequence, and all or part of a regulatory sequence such as a promoter The translation initiation sequence can be removed or replaced. Cut.

[0059] As used herein, "gene knock-in" refers to the process of adding a genetic function to a host cell. This results in increased levels of the encoded protein. As will be understood, this may include adding one or more additional copies of a gene to a host cell; or by modifying the regulatory components of endogenous genes to increase protein expression. This can be achieved in several ways, including by modifying the promoter, Adding a promoter, adding an enhancer, or modifying other gene expression sequences This can be achieved by:

[0060] The "β-2 microglobulin" or "β2M" or "B2M" protein consists of the following amino acid sequence: The human gene is referred to as the human β2M protein having the sequence and nucleic acid sequence; No. NC_000015.10:44711487-44718159.

[0061] The "CD47 protein" protein has the amino acid and nucleic acid sequences shown below. Refers to human CD47 protein; the human gene is located under accession number NC_000016.10:1086620 8-10941562.

[0062] "CIITA protein" refers to a human CIITA protein having the amino acid and nucleic acid sequences shown below. The human gene is identified by the accession number N C_000003.12:108043094-108094200.

[0063] "Wild type" in the context of a cell means a cell as found in nature. However, As used herein in the context of pluripotent stem cells, it refers to the nuclei that result in pluripotency. may contain acid alterations, but have not been subjected to the gene editing procedures of the present invention to achieve low immunogenicity. It also refers to iPSCs.

[0064] As used herein, "syngeneic" refers to a gene that is immunologically compatible (e.g., does not produce an immune response). , refers to the genetic similarity or identity between the host organism and the cell graft.

[0065] As used herein, "allogeneic" refers to a genetic relationship between the host organism and the cell graft, where an immune response is generated. This refers to the difference in

[0066] As used herein, "B2M- / -" refers to a diploid cell in which the B2M gene is deficient in both chromosomes. As described herein, this can be done in a variety of ways. This can be done.

[0067] As used herein, "CIITA- / -" refers to a condition in which the CIITA gene is absent in both chromosomes of a diploid cell. This means that the IL-15 gene is inactivated. As described herein, this can be accomplished in a variety of ways. It is possible to do so.

[0068] As used herein, "CD47 tg" (meaning "transgene") or "CD47+" refers to Some host cells possess at least one additional copy of the CD47 gene. This means that the chief cells express CD47.

[0069] "Oct polypeptide" refers to a naturally occurring member of the Octamer family of transcription factors; or any of their mutants that retain transcription factor activity, which is most closely related to the Related naturally occurring family members or naturally occurring family members is similar (at least Within 50%, 80%, or 90% of the activity of the target gene), and may further contain a transcription activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, Oct-10, Oct-11, Oct-12, Oct-13, Oct-14, Oct-15, Oct-16, Oct-17, Oct-18, Oct-19, Oct-20, Oct-21, Oct-22, Oct-23, Oct-24, Oct-25, Oct-26, Oct-27 Oct3 / 4 (herein referred to as "Oct4") is a POU domain. It contains a 150 amino acid sequence conserved among uric-86, Pit-1, Oct-1, Oct-2, and uric-86. (See Ryan, AK & Rosenfeld, MG, Genes Dev. 11:1207-1225 (1997) (The entire contents of which are incorporated herein by reference). In some embodiments, the variant is , as described above, or Genbank accession number NP-002692.2 (human Oct4) or naturally occurring Oct polypeptides such as those described in NP-038661.1 (mouse Oct4). At least 85% across their entire sequences compared to members of the Peptide family , 90%, or 95% amino acid sequence identity. Oct polypeptides (e.g., Oct3 / 4 or Oct4) may be of human, mouse, rat, bovine, porcine, or other animal origin. Generally, proteins of the same species as the cells being engineered are used together. The Oct polypeptide promotes induction of pluripotency in non-pluripotent cells. It may be a pluripotency factor that can

[0070] "Klf polypeptide" refers to a naturally occurring member of the family of Kruppel-like factors (Klfs). The amino acid sequence of the Drosophila embryo pattern regulator Kruppel is Zinc finger proteins containing amino acid sequences similar to the sequence of the transcription factor The term refers to any naturally occurring member of the family that possesses the same transcription factor activity as the most closely related member. Related naturally occurring family members or naturally occurring family members is similar to a polypeptide that contains at least the DNA binding domain of 50%, 80%, or 90% activity), and may further contain a transcription activation domain ( See Dang, D.T., Pevsner, J. & Yang, V.W., Cell Biol. 32:1103-1121 (2000). (The entire contents of which are incorporated herein by reference.) Exemplary Klf family members include Members include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11 , Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 are It has been found to be a factor capable of generating iPS cells in mice. Similarly, the related genes Klf1 and Klf5 also promote iPS cell proliferation in mice, albeit with reduced efficiency. We were able to produce cells (Nakagawa, et al., Nature Biotechnology 26:101-106 (200 7), which is incorporated herein by reference in its entirety. In some embodiments, the variant may be any of those described above or the variant described in Genbank Accession No. CAX16088. (mouse Klf4) or CAX14962 (human Klf4) Compared to members of the Klf polypeptide family, there are fewer The Klf polypeptides (e.g., For example, Klf1, Klf4, and Klf5) can be expressed in humans, mice, rats, cows, pigs, or other animals. Generally, the species of the cell being engineered is the same as the species of the cell. The Klf polypeptide may be a pluripotency factor. Expression of the gene or polypeptide promotes induction of pluripotency in a starting cell or population of starting cells. We can move forward.

[0071] "Myc polypeptide" refers to any naturally occurring member of the Myc family. (e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6:635-645 (20 05), which is incorporated herein by reference in its entirety. Naturally occurring family members or a small number of naturally occurring family members Similar transcription factor activity (at least in comparison with polypeptides containing a DNA-binding domain) The term also includes variants having activity within 50%, 80%, or 90% of the naturally occurring The present family members contain polypeptides that contain at least the DNA binding domain. Exemplary Myc polypeptides include, for example, Examples include c-Myc, N-Myc and L-Myc. In some embodiments, the variants are or Genbank accession number CAA25015 (human Myc). Compared to naturally occurring members of the Myc polypeptide family, such as those At least 85%, 90%, or 95% amino acid sequence identity across their entire sequences Myc polypeptides (e.g., c-Myc) are expressed in human, mouse, rat, bovine, porcine, and They may be of animal or other origin, generally from the same species as the engineered cells. (species) proteins together. Myc polypeptide is a pluripotency factor. There is.

[0072] "Sox polypeptides" are those that contain a high-mobility group (HMG) domain. Naturally occurring members of the SRY-related HMG box (Sox) transcription factors are characterized by Bar, or similar transcription factor activity compared to the closest related naturally occurring family (at least 50%, 80%, or 90% of the activity) It also reveals that at least the DNA-binding domain of the naturally occurring family members and may further include a transcription activation domain (e.g., Dang, See DT et al., Int. J. Biochem. Cell Biol.32:1103-1121 (2000). (The entirety of which is incorporated herein by reference.) Exemplary Sox polypeptides include, e.g., , Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, So These include Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 is related to Sox2 and It has been shown to generate iPS cells with similar efficiency and is mediated by the genes Sox3, Sox15, and Sox18. has also been shown to generate iPS cells, but somewhat less efficiently than Sox2 (Nakagawa, et al., Nature Biotechnology 26:101-106 (2007), which is incorporated by reference in its entirety. In some embodiments, the variants are as described above. or those described in Genbank accession number CAA83435 (human Sox2) Compared with naturally occurring members of the Sox polypeptide family, their full sequences have at least 85%, 90%, or 95% amino acid sequence identity across the string. The polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) may be human, mouse, or rat. The species of cells to be engineered (species) may be human, bovine, porcine, or other animal origin. ecies) together with proteins from the same species. Sox polypeptides are pluripotent As discussed herein, SOX2 protein may be a factor in the differentiation of iPSCs. The present invention finds particular use in making

[0073] As used herein, the term "differentiated, hypoimmunogenic pluripotent cells" or "differentiated HIP cells" or "d "HIP cells" are cells engineered to be hypoimmunogenic (e.g., knock-in of B2M and CIITA) knock-in of CD47 and CD48) into the cell type that is ultimately transplanted into the subject. It means differentiated iPS cells. Thus, for example, HIP cells are hepatocytes ("dHIP hepatocytes"). ), beta-like pancreatic cells or pancreatic islet organoids ("dHIP beta cells"), endothelial cells ("dHIP endothelial cells"). They can differentiate into various types of cells, such as

[0074] The term percent "identity" in the context of two or more nucleic acid or polypeptide sequences The sequence comparison algorithms described below (e.g., BLASTP and BLASTN) or those skilled in the art can be used. The best fit is determined using one of the available algorithms (or other algorithms) or by visual inspection. When compared and aligned for maximum match, a certain percentage of nucleotides or refers to two or more sequences or subsequences in which the amino acid residues are identical. The percent "identity" is determined over a region of the compared sequences (e.g., over a functional domain). be present throughout the entire length of the two sequences being compared. For sequence comparison, typically one sequence is used as a counter sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and control sequences Enter the above into the computer, specify subsequence coordinates if necessary, and run the sequence algorithm. The sequence comparison algorithm is then run according to the specified program parameters. Percent sequence identity of test sequences relative to control sequences based on program parameters Calculate unity.

[0075] Optimal alignment of sequences for comparison can be achieved using methods such as those described by Smith & Waterman et al. , Adv. Appl. Math. 2:482 (1981) by the local homology algorithm Needlema Homology alignment algorithm from N. & Wunsch, J. Mol. Biol. 48:443 (1970) Similarity to Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988) Implementing these algorithms on a computer using a search method (Wisconsin ·Wisconsin Genetics Software Package, GAP and BEST are included in the Genetics Computer Group, 575 Science Dr., Madison, Wis. FIT, FASTA, and TFASTA) or by visual inspection (see Ausubel et al. below). Generally, this can be done.

[0076] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is BLAST. The algorithm is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is available from the National Center for Biotechnology Information. -(the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov / )) It is publicly available through.

[0077] "Inhibitors," "activators," and "modulators" are agents that affect the function or expression of biologically relevant molecules. The term "modulator" includes both inhibitors and activators. , expression or activity of a target molecule In vitro and In vivo Identify using an assay It is possible.

[0078] "Inhibitors" are, for example, For example, agents that inhibit expression or bind to a target molecule or protein. These may partially or totally block stimulation or have protease inhibitor activity. They inactivate, desensitize, or downregulate the activity of the target protein described. Reduce, decrease, inhibit, etc. the activation of Modulators may be antagonists of the target molecule or protein. This may happen.

[0079] An "activator" is, for example, a compound that induces or activates the function or expression of a target molecule or protein. They are drugs that bind to target molecules and stimulate, enhance, or open up the activity of the target molecules. An activator may act as an agonist for a target molecule or protein. It may be a.

[0080] A "homolog" is a compound that is similar to or has a similar structure at the nucleotide sequence, peptide sequence, functional, or structural level. A homolog is a biologically active molecule that is similar to a molecule. A homolog has a certain percent identity to a reference sequence. Thus, in one embodiment, homologous sequences or derivatives may be used. The conductor sequences share at least 70 percent sequence identity. In certain embodiments, Homologous or derivative sequences share at least 80 or 85 percent sequence identity. In certain embodiments, a homologous or derivative sequence has at least 90 percent sequence identity. In certain embodiments, the homologous or derivative sequences share at least 95 percent In a more particular embodiment, the homologous or derived sequence shares 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. A homologous or derivative nucleic acid sequence is also These sequences are suitable for use under high stringency hybridization conditions. The nucleic acid sequence may also be defined by its ability to remain bound to a reference nucleic acid sequence under certain conditions. A homologue that is structurally or functionally similar to a reference molecule is a chemical derivative of the reference molecule. Detecting, producing and screening structural and functional homologs and derivatives Methods for this are known in the art.

[0081] "Hybridization" generally occurs when complementary strands are in an environment below their melting temperature. It depends on the ability of denatured DNA to reanneal when hybridized with a probe. The higher the desired degree of homology between the possible sequences, the greater the relative As a result, the higher relative temperatures make the reaction conditions more stringent. The lower temperature tends to result in a lower stringent state. Changing the stringency of the hybridization reaction For further details and explanation, see Ausubel et al., Current Protocols in Molecular Biolog y, Wiley Interscience Publishers (1995), the entire contents of which are incorporated herein by reference. It is incorporated into the fine print.

[0082] The "stringency" of a hybridization reaction is determined by those skilled in the art. can be easily determined and generally depends on the probe length, washing temperature, and salt concentration. Generally, a longer probe is required for proper annealing. require higher temperatures, while shorter probes require lower temperatures.

[0083] "Stringent conditions" or "high stringency conditions" as defined herein can be determined by the following conditions: (1) low ionic strength and high temperature for washing (e.g. For example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate. (2) using a denaturing agent such as formamide during hybridization; A solvent such as 50% (v / v) formamide / 0.1% Ficor with 0.1% bovine serum albumin may be used. 750 mM ethanol / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (Ph 6.5) (3) 5 Mm sodium chloride, 75 Mm sodium citrate and use at 42°C; 0% formamide, 5xSSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate Thorium (Ph 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated Salmon sperm DNA (50 μl / ml), 0.1% SDS, and 10% dextran sulfate in a solution of 42 Hybridization was performed overnight at 37 °C in 0.2x SSC (sodium chloride / sodium citrate). After washing for 10 min in 0.1× SSC containing EDTA at 55°C, the cells were incubated for 10 min in 0.1× SSC. Perform a stringent wash for 10 minutes.

[0084] Any maximum numerical limitation given throughout this specification shall not be construed as limiting the present invention as if it were any lower numerical limitation. including any lower numerical limitation, as if each lower numerical limitation were expressly written herein. Any minimum numerical limitation given throughout this specification is intended to be construed as if it were greater than or equal to the numerical limit. Any higher numerical limit will be expressly set forth herein, as if such higher numerical limit were expressly written herein. Any numerical range given throughout this specification will include limitations as though fully set forth herein. Such broader numerical ranges shall apply to all matters contained herein, as if such narrower numerical ranges were all expressly written herein. "Narrower" Numerical Ranges are inclusive of any narrower numerical ranges that fall within a given numerical range.

[0085] As used herein, the term "modification" refers to a molecule that physically distinguishes the modified molecule from the parent molecule. In one embodiment, the CD47, HSVtk, and The modified molecule is formed by amino acid changes in the EC-CD or iCasp9 mutant polypeptide. the corresponding parent molecule that has not been modified according to the methods described herein, such as a wild-type protein; Does not include naturally occurring mutant proteins or modifications of such mutant polypeptides. In another embodiment, the variant polypeptide is a variant It contains one or more modifications that distinguish the function of the polypeptide from that of the unmodified polypeptide. For example, amino acid changes in a variant polypeptide may affect its receptor binding profile. In other embodiments, the mutant polypeptide comprises a substitution, deletion, or insertion modification, or a combination thereof. In another embodiment, the mutant polypeptide is one or more modifications that increase its affinity for a polypeptide relative to the affinity of the unmodified polypeptide Includes.

[0086] In some embodiments, a variant polypeptide has one or more amino acid sequences different from the corresponding native or parent sequence. In certain embodiments, the mutant polypeptide comprises one, two, three or four substitutions, insertions, or deletions. 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.

[0087] In this specification, the term "episomal vector" refers to a vector that exists in the cytoplasm of a cell and replicates autonomously. It refers to a genetic vector that can be inserted into the genomic DNA of a host cell; Many episomal vectors are known in the art and include the following: Please see below.

[0088] A "knockout" in the context of a gene refers to a gene that is capable of expressing ... This means that the gene does not produce a functional protein product. As such, knockout involves the removal of all or part of a coding sequence, eliminating a functional protein. Introduce a frameshift mutation (a truncated sequence or a number) to prevent the production of a protein. and removing or modifying regulatory elements (e.g., promoters), The gene is not transcribed, and translation is inhibited by binding to mRNA, etc. Generally, the knockout occurs at the genomic DNA level. so that the progeny of said cell are also permanently knocked out.

[0089] In the context of a gene, a "knock-in" refers to a gene that is capable of expressing ... This means having a more functional protein that is active within the cell. As such, knock-ins can be achieved in a variety of ways, including by replacing regulatory elements. Alternatively, gene expression can be achieved by transfecting the endogenous gene with a constitutive promoter (e.g., by adding a constitutive promoter to the endogenous gene). However, typically, they contain at least one copy of a protein-encoding transgene (tg). This can be done in a variety of ways, such as by introducing the gene into the cell. Quinning technology results in extra copies of the transgene being integrated into the host cell.

[0090] VII. Cells of the Invention The present invention involves starting with wild-type cells and rendering them pluripotent (e.g. For example, one could generate induced pluripotent stem cells, or iPSCs, and then generate HIP cells from the iPSC population. The present invention provides compositions and methodologies for producing HIP cells.

[0091] A. Methodology for Genetic Modification The present invention generates both pluripotent cells and HIP cells. Exemplary techniques include methods for modifying nucleic acid sequences in cells or under cell-free conditions for the purpose of Recombination, knock-in, ZFNs (zinc finger nucleases), TALENs (transcriptional activation enzymes) CRISPR (clustered ordered space receptor nuclease) Interspaced short palindromic repeats (IPRs) / Cas9, and other site-specific nuclease technologies These techniques allow for double-stranded DNA breaks at the desired locus. These controlled double-strand breaks facilitate homologous recombination at specific gene loci. This process focuses on targeting specific sequences of nucleic acid molecules such as chromosomes. recognize and bind to that sequence and induce a double-stranded break in the nucleic acid molecule. The double-stranded break is targeted using an endonuclease, which induces mutations in the DNA that cause non-homologous end joining. The repair is done by either non-homologous end-joining (NHEJ) or homologous recombination (HR). can be.

[0092] As will be appreciated by those of skill in the art, iPSCs may be modified to be less immunogenic as outlined herein. Similar to modifying the pluripotent cells of the present invention, a number of different techniques can be used to modify the pluripotent cells of the present invention. There are times when I do.

[0093] In general, these techniques can be used individually or in combination. To generate these cells, we used viral techniques to knock in the function of CD47 (e.g., Using CRISPR to express active B2M and / or can reduce the expression of CIITA protein. Thus, one embodiment includes a CRISPR step to knock out B2M, followed by a CITIA step. The CRISPR step to knock out the gene, and the Len step to knock in the function of CD47. The final steps of the virus are utilized in the order of, but using different techniques and in a different order. These genes can be manipulated.

[0094] As discussed more fully below, transient expression of reprogramming genes is commonly used This is performed to generate / induce potent stem cells.

[0095] In one embodiment, the cell is transformed with CRISPR technology, as known in the art. Clustered Regularly Interspaced Short Palindromic Repeats / Cas (CRISPR) CRISPR is used to generate starting iPSCs or to generate iPSCs. It may be used to generate HIP cells from genomic DNA. There are a number of CRISPR-based techniques. See, e.g., Doudna and Charpentier, Science doi: See 10.1126 / science.1258096. CRISPR technology and kits are commercially available.

[0096] b. TALEN Technology In some embodiments, the HIP cells of the present invention express transcription activator-like enzymes. Efector nuclease ( T ranscription A ctivator- L ike E effector N ucleases (TALEN TALENs can be generated using the methodology of the TALENs described in the literature, which can bind to virtually any desired DNA sequence. A restriction enzyme combined with a nuclease engineered to cleave it. Kits are commercially available.

[0097] c. Zinc Finger Technology In one embodiment, the cells are It is engineered using Zn-F Zinc finger nucleases combine a zinc finger DNA binding domain with a DNA cleavage domain. This is an artificial restriction enzyme created by fusing the Zn finger domain with can be engineered to target a specific desired DNA sequence, thereby Link finger nucleases target unique sequences in complex genomes By utilizing endogenous DNA repair mechanisms, these agents Like RISPR and TALENs, they can be used to precisely modify the genomes of higher organisms. Cut.

[0098] d. Viral-based techniques to generate HIP cells of the present invention (as well as to optimize iPCSs) There are a wide variety of viral technologies that can be used to initially create Vectors that can be used include, but are not limited to, retroviral vectors, lentiviral vectors, and aviral vectors. These include the use of denovirus vectors and Sendai virus vectors. The episomal vectors used in the preparation are described below.

[0099] e. Down-regulation of genes using interfering RNA In another embodiment, HLA-specific The genes encoding proteins used in the offspring are down-regulated by RNAi technology. RNA interference (RNAi) is a process in which RNA molecules target and often degrade specific mRNA molecules. It is a process that inhibits gene expression by binding to two types of RNA molecules - Small interfering RNA (miRNA) and small interfering RNA (siRNA) are at the heart of RNA interference. They target It binds to mRNA molecules and increases or decreases their activity. RNAi helps cells defend themselves against parasitic nucleic acids such as transposons. also has an impact on.

[0100] The sdRNA molecule is a single fragment of asymmetric siRNAs that contains a guide (antisense) strand of 19-21 bases. They are a 5' phosphate, a 2' Ome or 2' F modified pyrimidine, and six 3' They also contain a 3'-linked sterol moiety, two phosphatidylcholines at the 3' position, It also contains a sense strand containing sulfothioate and 2' Ome modified pyrimidines. Both strands are of length Contains a 2' omega-purine with no more than three consecutive stretches of unmodified purines. NA is disclosed in U.S. Patent No. 8,796,443, the entirety of which is incorporated herein by reference. It is inserted.

[0101] All of these techniques can be used to generate recombinant nucleic acids, as outlined herein. In certain embodiments, the recombinant nucleic acid (the desired polypeptide) is either encoding a peptide, such as CD47, or encoding a disruption sequence. operably incorporating one or more regulatory nucleotide sequences in the expression construct. The regulatory nucleotide sequence will generally be appropriate for the host cell and the subject being treated. For a variety of host cells, a large number of suitable expression vectors and suitable Such regulatory sequences are known in the art. Typically, one or more regulatory nucleotides are The sequence includes, but is not limited to, a promoter sequence, a leader or a signal sequence. , ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancers. Constitutive promoters known in the art may include a promoter sequence or an activator sequence. Also contemplated are naturally occurring or inducible promoters. promoters that combine elements of two or more promoters, or hybrids that combine elements of two or more promoters The expression construct can be either a promoter or a The expression construct may be present on an episome, such as a plasmid, or the expression construct may be present on a chromosome. In certain embodiments, the expression vector may be inserted into a transformed host. In certain embodiments, the cell comprises at least one marker gene that allows the selection of the cell. A nucleic acid sequence encoding a mutant polypeptide operably linked to one regulatory sequence. As used herein, a regulatory sequence includes a promoter, a nucleic acid sequence, a nucleic acid sequence, and a nucleic acid sequence. In certain embodiments, the expression vector comprises a sequence, an enhancer, and other expression control elements. The selection of the host cell to be transformed, the mutant polypeptide to be expressed, the copy number of the vector, and The ability to control the copy number of the vector or other vector-encoded features such as antibiotic markers. The expression of the protein is designed to be selected.

[0102] Examples of suitable mammalian promoters include, for example, promoters from the following genes: Examples include hamster ubiquitin / S27a promoter (WO 97 / 15664), monkey vacuolar promoter Simian vacuolating virus 40 (SV40) early promoter, adenovirus Rous major late promoter, mouse metallothionein-I promoter, Rous sarcoma mouse Respiratory syncytial virus (RSV) long terminal repeat region, mouse mammary tumor virus promoter (mouse mammary tumor virus mary tumor virus (MMTV) promoter, Moloney murine leukemia virus long terminal repeat region, and the early promoter of the human cytomegalovirus (CMV). Examples of promoters are actin, immunoglobulin or heat shock promoters.

[0103] In a further embodiment, the promoter for use in a mammalian host cell is pox virus, fowlpox virus (UK Patent No. 2,211,500 published on July 5, 1989) No. 4), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus derived from the rovirus, hepatitis B virus, and simian virus 40 (SV40) In a further embodiment, a heterologous mammalian promoter is used. The actin promoter, immunoglobulin promoter, and heat shock promoter were The SV40 early and late promoters also contain the SV40 viral replication origin. It can be conveniently obtained as an SV40 restriction fragment. Fiers et al., Nature 273: 113-120 (197 8) Human cytomegalovirus immediate early promoter ) is conveniently obtained as a HindIII E restriction fragment. Greenaway, PJ et al., Gene 18: 355-360 (1982). The above references are incorporated by reference in their entireties.

[0104] B. Generation of Pluripotent Cells The present invention provides a method for producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to provide said pluripotent stem cells.

[0105] Mouse and human pluripotent stem cells (commonly called iPSCs; miPSCs for mouse cells, The generation of human pluripotent stem cells (hiPSCs) is generally known in the art. As will be appreciated by those of skill in the art, there are a variety of different methods for making iPCSs. The first induction was performed by transfecting mouse embryonic or adult fibroblasts with four transcription factors, Oct3 / 4, Sox2, and c-Myc. and Klf4 were introduced by virus; Takahashi and Yamanaka Cell 126:663-676 (200 6) (in its entirety and specifically for the techniques outlined therein, see Since then, many methods have been developed; see For more details, see Seki et al., World J. Stem Cells 7(1):116-125 (2015) and Lakshmipathy and Vermuri, ed., Methods in Molecular Biology: Pluripotent Stem Cells, Methods and See, e.g., J. Immunol. 2013, 2013, both in their entirety and in particular with regard to hiPSCs. For methods for preparing the is more clearly captured by

[0106] In general, iPSCs are generated by the transient expression of one or more “reprogramming factors” in host cells. These are typically produced by various methods, but are usually introduced using episomal vectors. Under certain conditions, a small number of cells are induced to become iPSCs (typically using a selection marker). (This step is less efficient because no stem cells are used.) Cells are "reprogrammed" to pluripotency. Once they become competent, they lose the episomal vector and use endogenous genes to produce the factor. The loss of this episomal vector results in a "zero footprint" The resulting cells are called "zero footprint" cells. These cells are designed to The fewer genetic modifications (in the system) the better, so this is desirable. Preferably, the resulting hiPSCs will have no permanent genetic modifications.

[0107] As will be appreciated by those skilled in the art, reprogramming agents that may be used or may be used The number of reprogramming factors can vary. In general, the fewer reprogramming factors used, the better the cells will grow. The efficiency of transformation of cells into a pluripotent state, as well as "pluripotency", decreases. If there are fewer binding factors, the cells will not be fully pluripotent and will only be able to differentiate into a few cell types. There is a possibility that cells that can

[0108] In some embodiments, a single reprogramming factor, OCT4, is used. In one embodiment, two reprogramming factors, OCT4 and KLF4, are used. In this embodiment, three reprogramming factors are used: OCT4, KLF4 and SOX2. In this state, four reprogramming factors, OCT4, KLF4, SOX2 and c-Myc, are used. In an embodiment of the invention, SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV4 0L T antigen, 5, 6 or 7 reprogramming factors selected from Cut.

[0109] In general, the genes for these reprogramming factors are known in the art and commercially available. For example, the vectors are delivered in episomal form. ThermoFisher / Invitrogen offers Zero Footprint Imaging for hiPSCs Sendai Virus Reprogramming Kit is available for the production of HIV-1. MoFisher also sells an EBNA-based system, see catalog number A14703. It refers to lighting.

[0110] In addition, there are many hiPSC lines available commercially, e.g., zero footprint, Gibco, a virus-free human iPSC cell line (登録商標) Episomal See hiPSC line, K18945 (see also Burridge et al., 2011, supra).

[0111] Generally, as known in the art, iPSCs can be reprogrammed as described herein. By transiently expressing ramifying factors, CD34+ umbilical cord blood cells, fibroblasts, etc. It is generated from non-pluripotent cells.

[0112] For example, omitting c-Myc and using only Oct3 / 4, Sox2, and Klf4 still allows the generation of iPSCs. Although they were successful in reprogramming the cells, the efficiency of reprogramming decreased.

[0113] In general, iPSCs express specific genes including KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc, and TCL1. These factors for the purposes of the present invention are novel and are characterized by the expression of certain factors. The increased expression may be via induction or regulation of an endogenous gene locus or via transgene expression. It can sometimes be derived from the expression of a child.

[0114] For example, mouse iPSCs cells have been reported by Diecke et al., Sci Rep. 2015, Jan. 28;5:8081 (doi:10 miPSCs can be produced in whole and in particular using the method described in the publication of the present application (p. 1038 / srep08081). The methods and reagents for preparing the same are incorporated herein by reference. See Idge et al., PLoS One, 2011 6(4):18293, in its entirety and in particular the summary therein. The methods described are incorporated herein by reference.

[0115] In some cases, as outlined herein, for example, a replica as generally shown in FIG. By assaying for programming factors or as outlined herein and in the Examples. The pluripotency of the cells is measured or confirmed by carrying out a differentiation reaction on the cells.

[0116] C. Generation of Low Immunogenic Pluripotent Cells The present invention relates to low immunogenic cells, as defined herein. The generation, manipulation, expansion, and transplantation of HIP cells from pluripotent cells is This is achieved by only three genetic changes, minimizing disruption of cellular activity while preserving the function of the cells. to immune silencing.

[0117] As discussed herein, in certain embodiments reduces the protein activity of MHC I and II (HLA I and II if the cells are human) This involves the deletion or abolishment of the genes that code for those components. In one embodiment, the coding sequence of the gene In another embodiment, the region or regulatory sequence is disrupted using CRISPR. The third change is the use of macrophage-mediated transcription factors such as CD47 to decrease gene translation. These are changes in genes that regulate susceptibility to phagocytosis, and are generally achieved using viral techniques. This is a "knock-in" of a gene that was previously present.

[0118] Using CRISPR to modify genes, and in some cases Cas9, which allows for highly efficient editing of cell lines. hiPSC cells containing the construct can be used. For example, Life Technologies The Human Episomal Cas9 iPSC cell line , see A33124.

[0119] 1. Reduction of HLA-I The HIP cells of the present invention have MHC I function (the cells are derived from human cells) In some cases, this involves reducing HLA I.

[0120] As will be appreciated by those skilled in the art, reducing function may involve removing a nucleic acid sequence from a gene. by inserting another sequence into the sequence, or by modifying the regulatory component of the nucleic acid, etc. For example, the entire coding region of the gene of interest may be cloned or cloned. frameshift mutations that can remove or replace a portion with a "nonsense" sequence It is possible to create a gene that can be used to remove or replace all or part of a regulatory sequence such as a promoter. The translation initiation sequence can be removed or replaced, etc.

[0121] As will be appreciated by those skilled in the art, in pluripotent cells, MHC I function (provided the cells are derived from human cells) may be achieved. In the case of the above, HLA I) is successfully reduced as described below. techniques such as those described herein; for example, FACS techniques using labeled antibodies that bind to said HLA complexes. For example, commercially available HLA-A, which binds to the alpha chain of human major histocompatibility HLA class I antigens A, B, C antibodies can be used to measure

[0122] In one embodiment, the reduction of HLA-I activity is achieved in pluripotent stem cells. This was done by disrupting the expression of the β-2 microglobulin gene in the mouse model and isolating its human sequence. This alteration is generally referred to herein as a gene "knockout," In the HIP cells of the present invention, both alleles of the host cell are involved. , the methods for destruction are the same.

[0123] Particularly useful embodiments use CRISPR technology to disrupt the gene. We then used CRISPR technology to introduce small deletions / insertions into the coding region of the gene. , preventing the production of a functional protein (often the result of a frameshift mutation) This results in the creation of a stop codon, a truncated, non-functional protein, and (

[0124] Thus, a useful technique involves the use of a coding sequence for the mouse B2M gene or the human B2M gene. The idea is to use CRISPR sequences designed to target the target gene. , disaggregate transfected iPSC cell cultures into single cells. Single Cells The clones were grown to full-sized colonies and were then analyzed to determine whether the aberrant sequence was present at the CRISPR cut site. The researchers then screen for the presence of both alleles and test for CRISPR editing. Select clones that are deleted. Such clones will not express B2M. , indeed shown by PCR and not expressing HLA-I by FACS analysis. This has been demonstrated (see, eg, Examples 1 and 6).

[0125] Assays for testing whether the B2M gene is inactivated are known, and In one embodiment, the assay comprises detecting a B2M protein. In another embodiment, the reverse is true. The presence of the inactivation modification was confirmed by transcription-polymerase chain reaction (rt-PCR). do.

[0126] The cells are further tested to confirm that the HLA I complex is not expressed on the cell surface. This can be confirmed by the binding of one or more HLA cell surface components as described above. It can be assayed by FACS analysis using antibodies.

[0127] Others have had poor results when trying to silence the B2M gene in both alleles. It is noteworthy that the results of this study were not only satisfactory, but also positive. For example, Gornalusse et al., Nature Biotech. oi / 10.1038 / nbt.3860).

[0128] 2. Reduction of HLA-II In addition to the reduction of HLA I, the HIP cells of the present invention also have MHC II function ( If the cells are derived from human cells, they also lack HLA II).

[0129] As will be appreciated by those skilled in the art, the reduction in function can be achieved by removing a nucleic acid sequence from a gene, Adding a nucleic acid sequence to a gene, disrupting the reading frame, or interrupting the sequence with another sequence This can be accomplished in a number of ways, including by altering the expression of the nucleic acid or by modifying the regulatory components of the nucleic acid. In one embodiment, all or part of the coding region of a gene of interest can be removed. In another embodiment, the promoter can be replaced with a "nonsense" sequence. Regulatory sequences such as the transcription start sequence can be removed or replaced, and translation initiation sequences can be removed or replaced. It is possible to do the following, etc.

[0130] In pluripotent cells or their derivatives, MHC II function (cells derived from human cells) In cases where the patient is diagnosed with HIV infection, it is important to confirm that the HLA protein has been successfully reduced by using antibodies against that protein. Western blotting, FACS technology, rt-PCR technology, and other techniques are used. It can be measured using the techniques of knowledge.

[0131] In one embodiment, the reduction in HLA-II activity is measured by altering the CIITA activity in pluripotent stem cells. This was achieved by disrupting expression of the ITA gene, the human sequence of which is presented herein. This modification is generally referred to herein as a gene "knockout" and is a method for producing the HIP cells of the present invention. In cells, both alleles of the host cell are transfected.

[0132] Assays for testing whether the CIITA gene has been inactivated are known. and described herein. In one embodiment, the assay comprises detecting a CIITA protein. Western blots of cell lysates probed with antibodies against The presence of the inactivating modification was confirmed by reverse transcription polymerase chain reaction (rt-PCR). I acknowledge.

[0133] The cells are further tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay can be performed as known in the art. (See, e.g., FIG. 21), generally as outlined below, human HLA class II HLA-D Western blot or F based on commercially available antibodies that bind to R, DP, and most DQ antigens This is done using either the ACS analysis.

[0134] A particularly useful embodiment uses CRISPR technology to disrupt the CIITA gene. The mouse Ciita gene or human CIITA, an essential transcription factor for MHC II molecules CRISPRs were designed to target the coding sequence of genes. After incubation, the transfected iPSC cell cultures are disaggregated into single cells. Cells were grown to full-sized colonies and then cloned into the CRISPR cut site with the abnormal sequence. Testing for successful CRISPR editing by screening for the presence of Clones carrying the deletion did not express CIITA as determined by PCR and did not express FA did not express MHC II / HLA-II as determined by CS analysis.

[0135] 3. Reduced phagocytosis. Knockout of B2M and CIITA is commonly used to reduce HLA I and II In addition to the reduction of MHC I and II, the HIP cells of the present invention inhibit phagocytosis by macrophages. The resulting HIPs have reduced susceptibility to phagocytosis and killing by NK cells. The cells are then transformed into immune macrophages and innate immune pathways by one or more CD47 transgenes. "Run away" from.

[0136] Increased CD47 In some embodiments, increased CD47 activity is observed in macrophages and NK cells. The decreased susceptibility to IL-1 activation was due to increased expression of CD47 on the HIP cell surface. This can be understood by those skilled in the art using "knock-in" or transgenic techniques. As will be appreciated, this can be accomplished in a number of ways. In some cases, increased expression of CD47 The addition of CD47 is due to one or more CD47 transgenes.

[0137] Thus, in some embodiments, one or more copies of the CD47 gene are inducible or constitutive. Alternatively, the gene may be added to HIP cells under the control of a specific promoter, the latter being preferred. In one embodiment, lentiviral constructs can be prepared as described herein or as described in the art. The CD47 gene is used as known in the art using suitable primers. It can be integrated into the genome of the host cell under the control of a promoter.

[0138] The HIP cell line was generated from B2M- / - CIITA- / - iPSCs. Cells containing the rRNA vector were selected using the blasticidin marker. 7 The gene sequence was synthesized and the DNA was inserted into the plasmid Lenti Cloned into virus pLenti6 / V5 (Thermo Fisher Science, Waltham, MA). did.

[0139] In some embodiments, expression of the CD47 gene is controlled by, for example, expression of the CD47 gene constitutively driven by an endogenous promoter. The endogenous CD47 gene was expressed by replacing the endogenous promoter with a constitutive promoter or a different inducible promoter. This can be increased by modifying the regulatory sequences of the This can be done using known techniques such as:

[0140] Once modified, sufficient CD47 expression was confirmed by Western blot analysis using an anti-CD47 antibody. - By known techniques such as blot, ELISA assay or FACS assay, as described in the examples. Generally, "sufficiency" in this context means that the HIP The expression of CD47 on the cell surface is increased, which supports killing by NK cells. The natural expression level on cells is that of their MHC I. When removed, it is too low to protect the cells from lysis by NK cells.

[0141] 4. Suicide Genes In some embodiments, the present invention provides a method for the detection of a "suicide gene" or "suicide switch." These provide low immunogenic pluripotent cells, including "low immunogenic pluripotent cells," which can be used to treat cancer if low immunogenic pluripotent cells are desired. "Anti-inflammatory agents" that can cause cells to die if they grow and divide in an undesirable way. The approach of elimination by "suicide genes" The probe is a protein that causes cell death only when activated by a specific compound. The suicide gene is contained in the gene transfer vector, which encodes a non-toxic enzyme. Alternatively, the gene encoding the enzyme may selectively convert the compound to a highly toxic metabolite. In some embodiments, the suicide gene is a hemoglobin-expressing The HSV thymidine kinase (HSV-tk) gene is the trigger for In another embodiment, the suicide gene is Escherichia coli cytosine deaminase (EC-C D) gene, the trigger of which is 5-fluorocytosine (5-FC) (Barese et al., ol. Therap. 20(10):1932-1943 (2012), Xu et al., Cell Res. 8:73-8 (1998), both (also incorporated herein by reference in its entirety).

[0142] In another embodiment, the suicide gene is an inducible caspase protein. Caspase proteins are proteins that can induce apoptosis. In one embodiment, the portion of the caspase protein comprises at least a portion of the sequence In a preferred embodiment, the inducible caspase protein is iCa sp9, which encodes human caspase 9 via a series of amino acids. Human FK506 binding protein (FKBP12) carrying the F36V mutation linked to the gene that encodes FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903 Therefore, the suicide function of iCasp9 in the present invention is mediated by a chemical inducer of dimerization. It is caused by the administration of ucer of dimerization (CID). In an embodiment, the CID is the small molecule drug AP1903. Dimerization inhibits apoptosis. Rapidly induced (WO2011146862;Stasi et al, N. Engl. J. Med 365;18 (2011);Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), respectively. (which is incorporated herein by reference in its entirety).

[0143] 5. Assay of HIP Phenotype and Retention of Pluripotency Once the HIP cells are generated, they These are, as outlined herein and in the Examples, due to their low immunogenicity, and / or for retaining pluripotency.

[0144] For example, low immunogenicity can be achieved using a number of techniques, such as those illustrated in Figures 13 and 15. These techniques include transplantation into an allogeneic host and evasion of the host immune system. and monitoring the proliferation of HIP cells (e.g., teratomas) in the presence of HIP cells. IP derivatives were transfected to express luciferase, The host cells can be tracked using bioluminescence imaging. T cell and / or B cell responses in the host animal were examined to determine whether HIP cells were responsible for the immune response in the host animal. Confirm that the antibody does not induce a reaction. T cell function is evaluated using Elispot and Elisa. , FACS, PCR, or mass cytometry (CYTOF). B cell responses or anti- The cell response is assessed using FACS or Luminex. evade innate immune responses (e.g., killing by NK cells as outlined in Figure 14) Lytic activity by NK cells may be assayed for their ability to inhibit NK cell proliferation and proliferation. As shown in Figure 15) In vitro or In vivo Evaluate based on:

[0145] Similarly, retention of pluripotency can be tested in a number of ways. The properties of certain pluripotency-specific factors are summarized herein and shown in FIG. Additionally or alternatively, the HIP cells are assayed for expression of They undergo differentiation into one or more cell types.

[0146] D. Preferred embodiments of the invention As the HIP cells or as differentiation products of the HIP cells When transplanted into an allogeneic host, such as a human patient, they exhibit pluripotency but do not elicit a host immune response. Provided herein are hypoimmunogenic pluripotent stem cells ("HIP cells") that are free of inflammatory cytokines.

[0147] In one embodiment, human pluripotent stem cells (hiPSCs) are derived from a) a B2M gene in each allele; b) disruption of the CIITA gene in each allele (e.g., CIITA- / -), and c) overexpression of the CD47 gene (CD47+, e.g., CD47-1 gene expression by introducing additional copies of the gene or activating the genome ), which renders the hiPSC population less immunogenic. In a preferred embodiment, the cells are non-immunogenic. HIP cells become non-immunogenic B2M- / - CIITA- / - CD47tg as described above, but can be used to inject IFN-γ into the blood. Therefore, In vivo By containing an inducible suicide gene that can be induced to kill cells at and is further modified.

[0148] E. Maintenance of HIP Cells Once generated, the HIP cells are used to maintain iPSCs. As is known, HIP cells can maintain an undifferentiated state. They are cultured on Matrigel using a medium that prevents differentiation and maintains pluripotency.

[0149] F. Differentiation of HIP Cells The present invention provides methods for differentiation of HIP cells into different cell types that can then be transplanted into a subject. As will be appreciated by those skilled in the art, methods of differentiation can be performed using known techniques. The cells are differentiated in suspension and the survival of the cells is then determined. To facilitate this, gels such as Matrigel, gelatin, or fibrin / thrombin forms may be used. Differentiation is generally indicated by the presence of cell-specific markers. The evaluation is assayed as known in the art.

[0150] In some embodiments, the HIP cells are differentiated into hepatocytes to ameliorate or prevent loss of hepatocyte function. addresses cirrhosis of the liver. There are many techniques that can be used to differentiate HIP cells into hepatocytes. be. For example, Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods M ol Biol 698:305-314 (2011), Si-Tayeb et al, Hepatology 51:297-305 (2010) and As see Gari et al., Stem Cell Rev (:493-504 (2013), all of which are incorporated herein by reference in their entirety. and specifically with respect to differentiation methods and reagents, are expressly incorporated herein by reference. Differentiated, as known in the art, includes, but is not limited to, albumin. Hepatocyte-associated and / or hepatocyte-associated proteins, including alpha-fetoprotein, fibrinogen, etc. or by assessing the presence of a particular marker. It regulates ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage. It can also be measured functionally, such as storage of acetylcholine.

[0151] In some embodiments, the HIP cells are cultured in cultured cells or pancreatic islet organelles for transplantation. The cell system is differentiated into inflammatory cytokines to combat type 1 diabetes mellitus (T1DM). This is a promising method for (see reference 1, incorporated herein by reference). In addition, Pagliuca et al. They reported that they were successful in differentiating these cells into β cells (doi / 10.106 / j.cell.2014.09.04 0, which describes the preparation of functional human β cells derived from human pluripotent stem cells in whole and in particular. The methods and reagents outlined for producing the compound on a large scale are incorporated herein by reference. Moreover, Vegas et al. have reported that human β cells can be generated from human pluripotent stem cells and then transplanted into host cells. It has been shown that encapsulation is used to avoid immune rejection by the host; (doi:10.103 8 / nm.4030, in whole and in particular, for the large-scale production of functional human β cells from human pluripotent stem cells. (which is incorporated herein by reference for the methods and reagents outlined for producing .

[0152] Differentiation generally refers to the acquisition of β cell-related proteins, including, but not limited to, insulin. By assessing the presence of related or specific markers, Differentiation can be confirmed by functional assays, such as measuring glucose metabolism. can also be measured in and, in their entirety and with respect to the biomarkers outlined in particular, are incorporated herein by reference. will be included).

[0153] Once dHIP beta cells have been generated, they can be cultured (as discussed herein) as a cell suspension. or in a gel matrix) in the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, Or they can be implanted into subcutaneous pouches.

[0154] In some embodiments, the HIP cells are differentiated into retinal pigment epithelium (RPE) to improve vision in the eye. Human pluripotent stem cells can be used to treat diseases that threaten the lives of many people. Kamao et al., Stem Cell Reports 2014:2 205-18 (in its entirety and in particular with regard to the methods and reagents outlined in relation to differentiation techniques and reagents) RPE Differentiation into endothelial cells has been achieved; see also Mandai et al., doi:10.1056 / NEJMoa1608368 Regarding the technology for producing RPE cell sheets and transplanting them into patients, is also incorporated in its entirety.

[0155] Differentiation is generally assessed by the presence of RPE-associated and / or specific markers. or by functional measurement, as known in the art. For example, Kamao et al., doi:10.1016 / j.stemcr.2013.12.007 (in its entirety and in particular the matrix outlined in the first paragraph of the Results section) (The disclosure of which is incorporated herein by reference.)

[0156] In some embodiments, the HIP cells are differentiated into cardiomyocytes to combat cardiovascular disease. Techniques for differentiating hiPSCs into cardiomyocytes are known in the art and are described in detail in the Examples. Differentiation is generally indicated by the expression of cardiomyocyte-associated markers or specific markers. By assessing the presence of a marker or by measuring its functionality, Assays can be performed as known in the art; see, e.g., Loh et al., doi:10.101 6 / j.cell.2016.06.001 (in its entirety and in particular for differentiation of stem cells, including cardiomyocytes, etc. (The method for the preparation of the fluororesin is incorporated herein by reference.)

[0157] In some embodiments, the HIP cells are differentiated into endothelial colony forming cells (ECFCs); To treat peripheral arterial disease by forming new blood vessels. See, e.g., Prasain et al., doi:10.1038 / nbt.3048 (in its entirety). and in particular, methods and reagents for generating endothelial cells from human pluripotent stem cells. (The same references are incorporated herein by reference with respect to the techniques for differentiation and transplantation.) Generally, by assessing the presence of endothelial cell-associated or specific markers, The assay may be performed by measuring the activity of the protein or by measuring the protein functionally, as known in the art. It is possible.

[0158] In some embodiments, the HIP cells are transformed into thyroid cells capable of secreting thyroid hormone. Differentiate into thyroid gland progenitor cells and thyroid follicular organoids to combat autoimmune thyroiditis. Techniques for differentiating oocytes into oocyte-like glandular cells are known in the art. See, e.g., Kurmann et al., doi:10.106 / j.stem.2015.09.004 (in its entirety and in particular, Regarding methods and reagents for producing thyroid cells from thyroid cancer, and regarding transplantation techniques, (Expressly incorporated herein by reference). Differentiation generally involves the differentiation of thyroid cell-associated markers. The protein can be measured functionally or by assessing the presence of a specific marker or The assay can be carried out by, as known in the art.

[0159] G. Transplantation of Differentiated HIP Cells As will be appreciated by those skilled in the art, the differentiated HIP derivatives may be Derivatives are available in a variety of formats, including those disclosed in the art, depending on both the cell type and the end use of these cells. Generally, the dHIP cells of the present invention are implanted intravenously or by injection. When transplanted to a specific location, the cells To prevent the cells from dispersing while retained, the gel matrix was May be suspended.

[0160] In order that the invention described herein may be more fully understood, the following examples are set forth. These examples are for illustrative purposes only and are not intended to limit the invention in any way. It should be understood that this is not to be construed as a EXAMPLES

[0161] (VIII. Examples) A. General Techniques 1. Generation of Mouse iPSCs Cells were obtained from Diecke et al., Sci Rep. 2015, Jan. 28;5:8081 (doi:10.1038 / srep08081). and relates generally and in particular to methods and reagents for producing said miPSCs. The same is incorporated herein by reference.

[0162] Mouse tail tip fibroblasts were cultured in collagenase type IV (Life Technologies, Inc.) Life Technologies, Grand Island, NY, USA The cells were dissociated and isolated using a 10% fetal bovine serum (FBS) supplemented with L-glutaminase (L-glutamate) and then incubated for 1 h in 10% FBS. glutamin, 4.5 g / L glucose, 100 U / mL penicillin, and 100 μg / mL streptomycin. We used Dulbecco's modified Eagle's medium (DMEM) containing isocyanin. The cells were then maintained in a humidified incubator at 37°C, 20% O2, and 5% CO2. Using the Neon Transfection system, four types of reprogramming We developed novel codon-optimized mini-transfectants expressing the transcription factors Oct4, KLF4, Sox2 and c-Myc. Using intron-plasmid (co-MIP) (10-12 μM DNA), 1 × 10 6 Mouse Wires After transfection, the fibroblasts were reprogrammed into MEFs. The cells were seeded on a feeder layer and supplemented with sodium butyrate (0.2 mM) and 50 μg / mL ascorbic acid. When ESC-like colonies appeared, the medium was changed to DMEM, 20 mL of fibroblast medium supplemented with 10% DMSO. % FBS, L-glutamine, non-essential amino acids (NEAA), β-mercaptoethanol, and 10 n The medium was changed to mouse iPSC medium containing 100 μg / mL leukemia inhibitory factor (LIF). The iPSCs were then transferred to 0.2% gelatin-coated plates for further expansion. Next, magnetic activated cell sorting (MACS) was used to The iPSCs were selected for the mouse pluripotency marker SSEA-1.

[0163] 2. Generation of human iPSCs hiPSCs were generated as described in Burridge et al., PLoS One, 2011 6(4):182. 93 (the entirety of which, and in particular with respect to the methods outlined therein, are incorporated herein by reference). The procedure was carried out as outlined in (http: / / www.ncbi.nlm.nih.gov / pubmed / 1234544).

[0164] Gibco (登録商標) Human episomal iPSC line (catalog no. A33124, Thermofish Thermo Fisher Scientific has developed three plasmids, 7 Species factors (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T This iPSC line was derived from CD34+ umbilical cord blood using an EBNA-based episomal system for the EBNA antigen. Since nothing is integrated into the genome from the reprogramming event, It is considered to be a footprint that does not include any of the reprogramming genes. It has been shown that

[0165] Gibco (登録商標) Human episomal iPSC lines were karyotypically normal and expressed the Oct4, S ox2, and Nanog (as shown by RT-PCR) and Oct4, SSEA4, TRA-1-60, and TRA-1-81 They endogenously express pluripotency markers such as pluripotency markers (indicated by ICC). Epigenetic profiling analysis confirmed that this episomal hiPSC line is a human They have been demonstrated to be molecularly indistinguishable from embryonic stem cell lines ( Burridge et al., 2011 ). In directed differentiation and teratoma analysis, these hiPSCs were able to differentiate into ectoderm, endoderm, and retained their differentiation potential toward mesodermal lineages (Burridge et al., 2011). In vitro, vascular, hematopoietic, nervous, and cardiac lineages were induced with robust efficiency (Burridge et al., 2003). t al., 2011).

[0166] 3. FACS analysis of surface molecules a. Detection of human HLA I surface molecules Human iPSCs, iCMs and iECs were seeded in 6-well plates and incubated with 100 ng / ml human IFNg (PeproTech, Roche, CA). Cells were harvested and stimulated with 100 mM NaCl (Peprotech, Rocket Hill, NJ). The APC-binding HLA-A, B, C antibody (clone G46_2.6, catalog number 562006, BD Biosciences) was used to Biosciences, San Jose, CA (clone G46_2.6, cat. No. 562006, BD BioS Sciences, San Jose, CA), or APC-conjugated IgG1 isotype control antibody (clone MOP HLA-A, B, and C antibodies were labeled with 100 mM MgCl, 100 mM NaCl, ... Binds specifically to the alpha chain of human major histocompatibility HLA class I antigens. Data Analysis The results were analyzed by flow cytometry (BD Biosciences) and analyzed by isotype. Expressed as percentage change relative to control.

[0167] 4. Detection of human HLA II surface molecules Human iPSCs, iCMs, and iECs were cultured in 6-well plates. and inoculated with 100 ng / ml human TNFα (PeproTech, Rocket Hill, NJ). The cells were then stimulated with Alexa-flour647-labeled HLA-D R, DP, DQ antibody (clone Tu3a, catalog number 563591, BD Biosciences, Sanno clone Tu3a, cat. No. 563591, BD BioSciences, San Jose, CA ) or Alexa-flour647-labeled IgG2a isotype control antibody (clone G155-178, Catalog The HLA-DR, DP, and DQ antibodies were labeled with human Major histocompatibility HLA class II Specific binding to HLA-DR, DP and most DQ antigens Data analysis was performed by flow cytometry (BD Biosciences). was expressed as percent change relative to isotype control.

[0168] 5. Detection of human CD47 surface molecule Human iPSCs, iCMs, and iECs were cultured in 6-well plates. The cells were seeded with 100 ng / ml human IFNg (PeproTech, Rocket Hill, NJ (P The cells were stimulated with PerCP-Cy5-conjugated CD47 (cloning, eprotech, Rocket Hill, NJ). B6H12, Catalog No. 561261, BD Biosciences, San Jose, CA ( clone B6H12, cat. No. 561261, BD BioSciences, San Jose, CA) or PerCP-Cy5 Conjugated IgG1 isotype control antibody (clone MOPC-21, catalog number 550795, BD Bio The B6H12 CD47 monoclonal antibody binds to the 42-52 kDa N-linked group. The data were analyzed by flow cytometry. The assay was performed using a ELISA kit (BD Biosciences), and the results were expressed as percentage changes relative to isotype controls. It is expressed as:

[0169] 6. Detection of mouse MHC I surface molecules MHC I surface molecules on miPSCs, miECs, miSMCs, and miCMs For detection of IL-1, cells were seeded on gelatin-coated 6-well plates and incubated at 100°C for 24 h. ng / ml of mouse IFNg (Peprotech, Rocket Hill, NJ) The cells were stimulated with PerCP-eFlour710-labeled MHCI antibody (Chloroform, Rocket Hill, NJ). AF6-88.5.5.3, Catalog No. 46-5958-82, eBiosciences, Santa Clara, CA State of California (clone AF6-88.5.5.3, cat. No. 46-5958-82, eBioscience, Santa Clara , CA)), or PerCP-eFlour710-labeled IgG2b isotype control antibody (clone eB149 / 10H 5, Catalog No. 46-4031-80, eBiosciences). The MHCI antibody was conjugated to H-2K b MHC class I reacts with alloantigens. Data analysis was performed by flow cytometry (BD Biosciences, The assay was performed by the National Institute of Integrative Medicine (NII) and results were expressed as percent change relative to isotype control.

[0170] 7. Detection of mouse MHC II surface molecules MHC II surface molecules on miPSCs, miECs, miSMCs, and miCMs For detecting molecules, cells were seeded on gelatin-coated 6-well plates. , 100 ng / ml mouse TNFα (PeproTech, Rocket Hill, NJ (PeproTech) The cells were stimulated with PerCP-eFlour710-conjugated MHC II antibody (MHC II, 100 mM NaCl, 100 mM NaCl, 100 mM Tris-Glu ... The clone M5 / 114.15.2 (catalog number 46-5321-82, eBioscience, Santa Cruz, CA) was used. Lara, CA (clone M5 / 114.15.2, cat. No. 46-5321-82, eBioscience, Santo a Clara, CA), or PerCP-eFlour710-labeled IgG2a / K isotype control antibody (clone eBM2a, Catalog No. 46-4724-80, eBiosciences). is a murine major histocompatibility complex class II antigen that is encoded by both the IA and IE subregions. Data were analyzed by flow cytometry (BD Biosciences, The assay was performed using a 3D ELISA (Figure 1) and the results were expressed as percent change relative to isotype control.

[0171] 8. Detection of mouse Cd47 surface molecules Cd47 surface molecules on miPSCs, miECs, miSMCs, and miCMs For detection of IL-1, cells were seeded on gelatin-coated 6-well plates and incubated at 100°C for 24 h. ng / ml of mouse IFNg (Peprotech, Rocket Hill, NJ) The cells were then stimulated with Alexa Fluor 647-labeled Cd47 antibody (Chloroform, Rocket Hill, NJ). miap301, Catalog No. 563584, BD Biosciences, San Jose, California (clone miap301, cat. No. 563584, BD BioSciences, San Jose, CA) or Alex a Fluor 647-labeled IgG2a / K isotype control antibody (clone R35-95, catalog no. 557 The Cd47 antibody was labeled with integrin-binding protein 690 (BD Biosciences). The data indicate that the IL-16 receptor agonist (IL-16) specifically binds to the extracellular domain of CD47, also known as an IAP. Analysis was performed by flow cytometry (BD Biosciences) and the results were analyzed by isotyping. The results were expressed as percentage change relative to the control.

[0172] 9. After allogeneic transplantation In vivo Determination of mouse cell morphology in allogeneic mice The rats were placed in a 250 mm cannula and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million (1 mio) cells (miPSC-derived cardiomyocytes (miCMs), miPSC-derived cardiomyocytes) in 1 μl of 0.9% saline were cultured at 4 °C for 1 h. miPSC-derived smooth muscle cells (miSMC) or miPSC-derived endothelial cells (miEC) were cultured at 250 1 μl of BD Matrigel High Concentration (1:1; BD The mice were injected subcutaneously into the lower back using a 23-G syringe (IoScience). After 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks, the Matrigel plugs were excised and reconstituted with 4% paraformaldehyde. The tissue was fixed in 1% glutenaldehyde and 1% glutenaldehyde for 24 hours, then dehydrated and paraffin-filled. Sections of 5 μm thickness were cut and stained with hematoxylin and eosin (HE). Stained with.

[0173] 10. After allogeneic transplantation In vivo Determination of human cell morphology in humanized NSG-SGM3 mice The rat was placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). ZVAD (100 mM, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl Le Ketone, Calbiochem), Bcl-XL BH4 (cell-permeable TAT peptide, 5 0 nM, Calbiochem), cyclosporine A (200 nM, Sigma), IGF-1 (100 ng / ml, Peptide in 250 μl of 0.9% saline containing 100 mM erythritol (Rotec) and pinacidil (50 mM, Sigma). 1 mio of cells (hiPSC-derived cardiomyocytes (hiCMs) or hiPSC-derived endothelial cells (hi 250 μl of BD Matrigel High Concentration ) (1:1; BD Biosciences) and injected into the lower back of the mouse using a 23-G syringe. The Matrigel plugs were then excised 2, 4, 6, 8, 10, and 12 weeks after implantation. The tissue was then fixed in 4% paraformaldehyde and 1% glutenaldehyde for 24 hours, followed by decontamination. The tissue was washed with water and embedded in paraffin. Five-μm-thick sections were cut and stained with hematoxylin and Stained with eosin (HE).

[0174] B. Example 1: β-2 microglobulin knockout pluripotent cells in a mouse model <Creation of artificial pluripotent cells>: Hypoimmune pluripotent cells were created in a mouse model. Human hypoimmune pluripotent cells are another embodiment generated using the strategies described herein. do.

[0175] Mouse induced pluripotent stem cells (miPSCs) were generated from C57BL / 6 fibroblasts. CF1 mouse embryonic fibroblast (MEF, Applied Stem Cells, Inc., CA) ) was thawed and resuspended in 10% heat-inactivated fetal calf serum (FCS hi), 1% MEM-NEAA, and 1% Penn-S Pen Strep (Thermo Fisher Scientific-Gibco, Waltham DMEM + GlutaMax 31966 (Gibco, Grand Rapids, MA) supplemented with MEF feeder cells were maintained in 100% confluent medium (Eland, NY). After monolayer formation, the cells were cultured in 15% KO serum replacement, 1% MEM-NEAA, 1% Pen-Strep (Thermo Fisher Scientific) and 1% MEM-NEAA. Fisher Scientific-Gibco), 1× β-mercaptoethanol and 100 μg / ml KO with LIF (Millipore, Billerica, MA) miPSCs were grown on MEFs in DMEM 10829. Cells were maintained in 10 cm dishes. The medium was changed daily and cultured with 0.05% trypsin-EDTA (Thermo Fisher Gibco). Cells were passaged every 2–3 days. miPSCs were cultured in standard medium without feeders. The MycoAlert kit (Lonza, Cologne, Germany) was used to culture the cells on 100% lysine 100% cellulose (Millipore). Cell cultures were routinely screened for mycoplasma infection.

[0176] <Mouse>: BALB / c (BALB / c AnNCr1, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c Nude (BALB / c NU / NU, CAnN.CgFoxn1 <nu> / Crl, H2d) and Scid Beige (CBySmn.CB17 -Prkdcscid / J) (all 6-12 weeks old) were used as recipients for the different assays ( All mice were 6–12 weeks old. Mice were cultured at Charles River Laboratories (Sulzfeld, Germany). ) (Charles River Laboratories, Sulzfeld, Germany) and were kept in compliance with the Animal Care Guidelines. Humane care in accordance with the Guide for the Principles of Laboratory Animals The animal experiments were carried out in accordance with the Hamburg Office for Health and Consumer Protection (Amt fur Gesundheit und The study was approved by the European Commission (European Commission) and carried out in accordance with regional and EU guidelines.

[0177] <Confirmation of pluripotency>: Pluripotency was confirmed by rtPCR. RNA was extracted using a ELISA kit (Thermo Fisher Scientific). A DNase I step was included to remove any genomic DNA remaining. Applied Biosystems (登 録商標) cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit. No reverse transcriptase A (no RT) control was also generated from all RNA samples. AmpliTaq Gold 360 Master M ix (Thermo Fisher Scientific-Applied Biosystems, W A gene-specific primer was used to amplify the target sequence using a genomic DNA primer (Bloomberg, MA, USA). PCR reactions were visualized on a 2% agarose gel. A positive control amplifying a constitutively expressed housekeeping gene (Actb) encoding The primer set was inserted. The results are shown in Figure 2. Pluripotency markers Nanog, Oct4, Sox2, Esrrb, Tbx3, and Tcl1 were detected by rtPCR in miPSC cells, but not in parental fibers. It was not detected in blast cells.

[0178] Pluripotency was also tested by immunofluorescence. miPSCs were seeded in 24-well dishes and After 48 hours, the cells were processed for RT-PCR and immunocytochemistry (ICC) analysis. The Image-iT Fixation / Permeabilization Kit (Thermo Fisher Scientific, Cells were fixed, permeabilized, and blocked using ELISA kit (Waltham, MA). The cells were stained with primary antibodies against Sox2 and Oct4 at 4°C overnight. After washing several times, Afterwards, the cells were incubated with AlexaFluor 488 secondary antibody and NucBlue Fixed Cell ReadyProbes Reage. The samples were incubated with nt (Thermo Fisher Scientific). The cells were imaged using a fluorescent microscope and were positive for Sox2 and Oct4. Data not shown.

[0179] Figure 3 shows that pluripotency was further confirmed by functional assays. 6 miPSC cells The recipient C57BL / 6 (syngeneic), BALB / c (allogeneic), and BALB / c nude (allogeneic) mice were The mice were injected into the thigh muscles of Scid beige (immunodeficient but T cell deficient) and Scid beige (immunodeficient) mice. Teratomas were formed in all mice except for the responsive allogeneic BALB / c mice.

[0180] <β-2 microglobulin knockout>: Knockout of the B2m gene using CRISPR technology The coding sequence of the mouse β-2-microglobulin (B2m) gene was targeted. To do this, follow the kit instructions (GeneArt CRISPR Nuclease Vector Kit). (Thermo Fisher Scientific, Waltham, Massachusetts) , annealing the CRISPR sequence 5'-TTCGGCTTCCCATTCTCCGG(TGG)-3', and Cas9 The expression cassette was then ligated into an all-in-one (AIO) vector (which was functional but not Another CRISPR that was less effective was 5'-GTATACTCACGCCACCCAC(CGG)-3' and 5'-GTATACTCACGCCACCCAC(CGG)-3'. The ion channel was '-GGCGTATGTATCAGTCTCAG(TGG)-3'. Two 1200V pulses of 20ms duration were The AIO vector was transfected into miPSCs using Neon electroporation in vitro. The transfected iPSC cultures were incubated with 0.05% trypsin (Gibco) for 1 h. The cells were then dissociated into single cells using the ELISA kit and their forward and side scatter emissions were then measured. To selectively gate and eliminate clumps and debris, a FACSAria (商標) Cell sorter (BD Biosciences, Franklin Lakes, NJ) Single cells were grown to full-sized colonies and then cultured at 4°C for 10 min. Testing CRISPR editing by screening for the presence of abnormal sequences at RISPR cut sites Briefly, AmpliTaq Gold Mastermix (Thermo Fisher Scientific) was used. (Dot Biosystems, Waltham, MA) and primer B2m gDNA:F: 5'-CTGGATCAGACATATGTGTTGGGA-3', R: 5'-GCAAAGCAGTTTTAAGTCCACACAG-3' The target sequence was then amplified by PCR.

[0181] After cleaning up the PCR products obtained (PureLink (登録商標) Pro 96 Pro Purifica tion Kit, Thermo Fisher Scientific, Waltham, Massachusetts. Ion Personal Genome Machine (PGM) (商標) , Thermo Fisher Scientific Sanger sequencing was performed using a genomic DNA sequencing kit (Tiffany's). ), i.e., a 250 bp region of the B2m gene, was sequenced using primers - B2m gDNA PGM: F: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3' and R: 5'- GGATT PCR amplification was performed using the sequence TCAATGTGAGGCGGGT-3'.

[0182] The PCR products were purified as described above and purified using the Ion PGM Hi-Q template kit (ThermoFusion). The experiments were performed using Ion 318 (商標) Chip Kit v2 (Thermo Fisher Scientific) was used to measure the Ion PGM (商標) Sis Analysis of pluripotency was performed again.

[0183] As can be seen in Figure 4, expression of β-2-microglobulin was knocked out in miPSC cells. MHC-1 expression was not induced by IFN-γ stimulation (right panel). As a control, parental miPSC cells were stimulated with IFN-γ (left panel).

[0184] C. Example 2: Generation of β-2 microglobulin / Ciita double knockout pluripotent cells To further knock out the iita gene, we used CRIPSR technology. To target the coding sequence of the gene, use the GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher, Waltham, Massachusetts (State of New York), which anneals the CRISPR sequence 5'- GGTCCATCTGGTCATAGAGG (CGG)-3'. The vector was then ligated into an all-in-one (AIO) vector containing a Cas9 expression cassette. miPSCs were transfected with the AIO vector using the same conditions as for (a). Transfected iPSC cultures were incubated with 0.05% trypsin (Thermo Fisher-Gibco) for 1 h. ) to dissociate into single cells, and then measure forward and side scatter emission. To eliminate clumps and debris by selectively gating the output, FACSA was used. ria (商標) Cell sorter (BD Biosciences, Franklin Lakes, New Zealand) Single cells were grown to full-sized colonies and then selected using the CRISPR editing was attempted by screening for the presence of abnormal sequences at the CRISPR cut site. Briefly, AmpliTaq Gold Mastermix (Thermo Fisher Scientific, Ido Biosystems, Darmstadt, Germany) and primers Ciita gDNA:F: 5 R: 5'-CCCCCAGAACGATGAGCTT-3', R: 5'-TGCAGAAGTCCTGAGAAGGCC-3', and The target sequence was amplified using the following PCR product. After cleaning up the PCR product (PureLink ( 登録商標) Pro 96 Pro Purification Kit, Thermo Fisher Scientific The DNA was then subjected to Sanger sequencing at 100 bp (Kanagawa University, Waltham, MA). Using DNA sequence chromatograms, we confirmed the presence of abnormal sequences at the CRISPR cleavage sites. The edited clones were identified by qPCR. Indel sizes were calculated using the TIDE tool. To confirm the pluripotent status of the cells, PCR and ICC were performed again.

[0185] Figure 5 shows that miPSC / β-2-microglobulin / Ciita double knockout MHC-II could not be induced to express MHC-II by TNF-α. Ta.

[0186] D. Example 3: Generation of β-2 microglobulin / Ciita double knockout-Cd47+ pluripotent cells The Cd47 expression vector was introduced into the B2m / Ciita double knockout miPSCs prepared above. The vector was then transformed with a lentivirus carrying the antibiotic resistance cassette blasticidin. The Cd47 gene sequence was synthesized and the DNA was then transfected into blastocytes. The plasmid Lentivirus pLenti6 / V5 (Thermo Fisher Scientific, Washington, DC) contains a 5'-receptor resistance marker. The clones were cloned into a plasmid (Lucifer, Massachusetts) and subjected to Sanger sequencing. It was confirmed that no mutations occurred. 7 Lentivirus at stock titer in TU / ml The recombinant vector was then transformed into 2 × 10 5 B2m-KO / Ciita Double Knockout miP SCs were transduced at an MOI of 1:10 and expanded on blasticidin-resistant MEF cells for 72 h. followed by antibiotic selection with 12.5 μg / ml blasticidin for 7 days. Cd47 mRNA was amplified by RT-qPCR, and Cd47 was analyzed by flow cytometry. The antibiotic-selected pools were then tested by detecting Cd47 expression. Once confirmed, the cells were expanded and subjected to pluripotency assays.

[0187] FIG. 6A shows the β-2-microglobulin / Ciita double knockout (iPS hypo Cells) Figure 6B shows that the expression of Cd47 was increased in allogeneic BALB / c mice. C57BL / 6 iPS cells were cultured under / c conditions. hypo The cells survive, but their parent iPS cells do not. This groundbreaking result shows that hypoimmune pluripotent cells can be transformed into non-hypimmune pluripotent cells. It has been shown that they survive when transplanted into hosts that are incompatible with them.

[0188] E. Differentiation of mouse cells from mHIP cells <pancreatic islet cells>: The mHIP cells were cultured according to the method of Liu et al., Exp. Diabetes Res 2012:201295 (doi:10.1155 / 2012 / 201295), incorporated herein by reference. and is hereby incorporated herein with particular reference to the differentiation techniques outlined therein) iPS cells were differentiated into pancreatic islet cells using a technique adapted from The cells were transferred to a flask for 30 min to remove the feeder layer and were then supplemented with 2 mM glutamine, 100 μM non-essential amino acids. Supplemented with 10 mM acetylcholinesterase, 10 ng / mL activin A, 10 mM nicotinamide, and 1 μg / mL laminin. 1 × 10 cells per well in DMEM / F-12 medium with 10% FBS. 6 Collagen I in cells ES-D3 cells were then seeded onto the coated plate. Essential amino acids, 10 ng / mL activin A, 10 mM nicotinamide, 25 μg / mL insulin, and placed in DMEM / F-12 medium with 2% FBS supplemented with 1 μg / mL laminin for 6 days.

[0189] <Neural stem cells>: The mHIP cells were cultured in a 30-well plate using a 30-well platelet-derived 1 ... 286 (which is incorporated herein by reference, and in particular with respect to the differentiation techniques outlined therein). The cells were differentiated into neurons using a technique adapted from the method described in the literature (incorporated herein). To begin the layering protocol, ES cells were cultured in serum-free medium ESGRO Complete Clone Grade High density (1.5 x 10 5 After 24 hours, ES cells were Gently dissociate the cells into pieces and place in RHB-A or N2B27 medium (Stem Cell Sciences, Inc.). mCell Science Inc.)), 1×10 4 cells / / cm 2 0.1% (v / v) gelatin coating The cells were seeded on tissue culture plastic and the medium was changed every other day. The cells were then dissociated and cultured in a 30-well plate with 5 ng / ml mouse bFGF (Peprotech). ) on laminin-coated tissue culture plastic. 4 cells / cm 2 From this point on, cells were reseeded every 4 days under the same conditions and the medium was changed to The medium was replaced daily and cultured for a total of 20 days. The number of differentiating neurons at each time point was quantified. To culture the laminin-coated glass coverslips in 24-well Nunc plates, The cells were seeded on the glass. Two days after seeding, the medium was changed to RHB-A:Neurobasal:B27 mixture (1:1). :1:0.02) to allow differentiated neurons to survive better.

[0190] <Smooth muscle cells>: The mHIP cells were purified by Huang et al., Biochem Biophys Res Commun 2006: 351(2)321-7 (incorporated herein by reference, and in particular the differentiation techniques outlined therein). The cells were differentiated into SM cells using a technique adapted from the method described in the present application (incorporated herein). The resuspended iPSCs were plated onto a 6-well gelatin-coated plastic petri dish. 2 million cells (2 mio) per well on a 30-well plate (Falcon, Becton Dickinson). cells, 10 μM atRA in 2 ml of differentiation medium at 37°C, 5% CO 2 The cells were cultured in the above-mentioned manner. The differentiation medium was prepared as follows: DMEM, 15% fetal bovine serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% The composition was prepared with non-essential amino acids, penicillin, and streptomycin. The culture was continued for 10 days with daily changes of medium.

[0191] From day 11, the differentiation medium was changed to Knockout DMEM, 15% Knockout serum replacement, 2 ml M L-glutamine, 1 mM MTG, 1 % non-essential amino acids, penicillin, and streptomycin The culture was continued for another 10 days with daily changes of serum-free medium. I did.

[0192] <Myocardial cells>: The mHIP cells were purified by Kattman et al., Cell Stem Cell 8:228-240 (2011) (Incorporated herein by reference, and in particular the differentiation techniques outlined therein. The cells were then differentiated into CM cells using a technique adapted from the previously described method (incorporated into the technical description).

[0193] Endothelial cells: The mHIP cells were differentiated into endothelial cells as previously described.

[0194] F. Example 4: Allogeneic transplantation of HIP cell derivatives results in fully immunocompetent recipients a. Mice: BALB / c (BALB / c AnNCr1, H 2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU, CAnN.CgFoxn1 <nu> / Crl, H2d) and Scid beige (CBySmn.CB17-Prkdcscid / J) (all 6-12 weeks old), They were used as recipients for the different assays (all aged 6-12 weeks). Animals per experimental group The numbers are shown in each figure. Mice were obtained from Charles River Laboratories (Sulzfeld, Germany). The animals were purchased from Charles River Laboratories (Sulzfeld, Germany) and kept in compliance with the guidelines for laboratory animal guidelines. They received humane care in accordance with the Guide for the Principles of Laboratory Animals. Animal testing is carried out in accordance with the regulations of the Hamburg Office for Health and Consumer Protection (Amt fur Gesundheit und V The study was approved by the European Commission (Eurbraucherschutz) and carried out in accordance with regional and EU guidelines.

[0195] b. Pluripotency analysis by RT-PCR and IF: miPSCs were seeded in 24-well plates and After 48 hours, the cells were processed for RT-PCR and immunofluorescence (IF) analysis. Image-iT (商標) Fixation / Permeabilization Kit (Thermo Fisher catalog number, R37602 The cells were fixed, permeabilized, and blocked with 100 μg / mL of ... The sections were stained with primary antibodies against ct4 and alkaline phosphatase at 4°C overnight. Afterwards, the cells were incubated with AlexaFluor 488 secondary antibody and NucBlue Fixed Cell ReadyProbes Reage. nt (all Thermo Fisher Scientific). The stained cells were imaged using a fluorescent microscope.

[0196] For RT-PCR, PureLink (商標) RNA Mini Kit (Thermo Fisher catalog no. RNA was extracted using a 50 mL aliquot of 1000 ng / ml PBS (No. 12183018A). A Nase I step was added. Applied Biosystems (登録商標) High-Capacity cDNA Reverse Sequence A no reverse transcriptase (no RT) control was also performed. AmpliTaq Gol was prepared from the sample. (登録商標) 360 Master Mix (Thermo Fisher (Cat. No. 4398876) to amplify the target sequence using gene-specific primers The PCR reactions were visualized on a 2% agarose gel. A positive control probe amplifying a constitutively expressed housekeeping gene (Actb) encoding I put in the Rhymer set.

[0197] c. Gene editing of mouse iPSCs: miPSCs underwent three gene editing steps. In the first step, we used CRISPR to target the coding sequence of the mouse B2m gene. The resulting vector was annealed and ligated to a vector containing a Cas9 expression cassette. The transfected miPSCs were dissociated into single cells and then dissociated into colonies. The clones were then grown, sequenced, and tested for homogeneity. In a second step, we transduced these B2m- / - miPSCs with the master receptor for MHC II molecules. The mice were transfected with vectors containing CRISPRs targeting the Ciita modulator. The expanded single-cell colonies were sequenced to identify abnormal sequences at the CRISPR cut sites. In the third step, B2m- / - Ciita- / - clones were identified through the presence of The Cd47 gene sequence was synthesized and the DNA was inserted into a blasticidin-resistant plasmid. The lentivirus was cloned into B2m- / - Ciita- / - miPSCs expressing blasticidin. The antibiotic-selected pool was then transfected and grown in the presence of Cd47 overexpression. We tested the expression of B2m- / - Ciita- / - Cd47 tg miPSCs and expanded them. In contrast, wt miPSCs showed high MHC I expression and low but detectable MHC II expression. In the miPSC line generated from wt miPSCs, expression of Cd47 was observed, and very little Cd47 expression was observed. The absence of MHC I expression, MHC II expression, and overexpression of Cd47 were confirmed in all The engineered miPSC lines were tested for pluripotency. / - Ciita- / - Cd47 tg miPSCs and that these cells are derived from all three germ layers. It was confirmed that these cells have the potential to form the new type of cells.

[0198] d. Generation of B2m- / - miPSCs: CRIPSR technology was used to knock out the B2m gene. The mouse beta-2-microglobulin (B2m) gene coding sequence was targeted To do this, follow the kit instructions (GeneArt CRISPR Nuclease Vector Kit , Thermo Fisher Scientific, Waltham, MA), CRISPR sequence 5'- TTCGG CTTCCCATTCTCCGG(TGG)-3' and the oligonucleotide containing the Cas9 expression cassette. The cells were then coupled to an Al-I-O (AIO) vector. Two 1200V pulses of 20ms duration were administered. The AIO vector was transfected into miPSCs using Neon electroporation. The transfected iPSC cultures were then cultured using 0.05% trypsin (Gibco). The cells were then dissociated into single cells and selectively analyzed for forward and side scatter emission. 2. Use a FACSAria Cell Gating System to eliminate clumps and debris by gating on Cell Count. Sorter (BD Biosciences, Franklin Lakes, NJ) Single cells were grown to full-sized colonies and then transfected with CRISPR cut sites. To test for CRISPR editing, we screened for the presence of abnormal sequences in the For the purpose of explanation, AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) was used. , Germany) and primers B2m gDNA F: 5'-CTGGATCAGACATATGTGTTGGGA-3', R: 5'- The target sequence was amplified by PCR using the following sequence: GCAAAGCAGTTTTAAGTCCACACAG-3' After cleaning up the PCR products (PureLink (登録商標) Pro 96 Pro Purificati The clones were analyzed by Sanger sequencing using the ELISA Kit (Thermo Fisher Scientific). We used Ion Personal Genome Machine (PGM) sequencing to identify the geneity of A 250 bp region of the B2m gene was amplified using primer B2m gDNA PGM F: 5'-TTTTCAAAATGTG GGTAGACTTTGG-3' and R: 5'- GGATTTCAATGTGAGGCGGGT-3' were used for PCR amplification. The PCR products were purified as previously described and purified using the Ion PGM Hi-Q Template Kit. The experiment was performed using Ion 318 (商標) Chip Kit v2 Using Ion PGM (商標) The study was carried out using a system that can analyze pluripotency. The analysis was performed again.

[0199] As previously reported in the field, there was no observed decrease in the proliferation rate or differentiation capacity of B2m- / - iPSCs. It wasn't noticed.

[0200] e. Generation of B2m- / - and Ciita- / - miPSCs to further knock out the Ciita gene To target the coding sequence of the mouse Ciita gene, we used CRIPSR technology. The vector was then incubated with 100% CRISPR DNA in 200 mL of 100% PBS according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Ser. (MO Fisher, Waltham, MA), CRISPR sequence 5'- GGTCCATCTGG TCATAGAGG (CGG)-3' was annealed and the allele containing the Cas9 expression cassette was inserted. The miPSCs were pre-transfected using the same conditions as for B2m-KO. The transfected miPSC cultures were cultured at 0.05 The cells were dissociated into single cells using trypsin (Gibco), and then The two masses and debris were isolated by selectively gating the forward and side-scattered emission. To remove the spheroids, a FACSAria cell sorter (BD Biosciences, Franklin Ray, CA) was used. Single cells were grown to full-sized colonies. The clones were then propagated and screened for the presence of aberrant sequences at the CRISPR cut sites. ISPR editing was tested. Briefly, AmpliTaq Gold Mastermix (Applied Biosciences) was used. Osystem, Darmstadt, Germany) and primer Ciita gDNA:F: 5'-CCCCCAGA ACGATGAGCTT-3', R: 5'-TGCAGAAGTCCTGAGAAGGCC-3', The target sequence was amplified. The PCR products were cleaned up (PureLink (登録商標) P ro 96 Pro Purification Kit, Thermo Fisher Scientific), and Sanger sequencing Next, DNA sequence chromatograms were used to identify the presence of aberrant sequences at the CRISPR cleavage sites. Edited clones were identified through the presence of indels. Indel sizes were analyzed using the TIDE tool. PCR and ICC were performed again to confirm the pluripotent status of the cells. .

[0201] f. Generation of B2m- / - Ciita- / - Cd47 tg miPSCs: antibiotic resistance cassette-mediated blastocytic differentiation Antibiotic resistance after lentiviral delivery of a Cd47 expression vector containing the gene By performing selection using the above method, the cell lines B2m-KO, Ciita-KO and Cd47-tg iPSC were generated. The Cd47 gene sequence was synthesized and the DNA was inserted into a plasmid containing blasticidin resistance. Lentivirus was cloned into pLenti6 / V5 (Thermo Fisher). Sanger Sequence Sequencing was performed to confirm that no mutations were present. 7 Stock potency in TU / ml Lentivirus was produced at a titer of 2 × 10 5 B2m- / - Ciita- / - miPSCs at an MOI of 1:10 The cells were transduced at a ratio of 1:1 and grown on blasticidin-resistant MEF cells for 72 h, followed by incubation at 7 Antibiotic selection was performed using 12.5 μg / ml blasticidin for 3 days. By amplifying RT-qPCR and detecting Cd47 by flow cytometry Therefore, we tested the antibiotic-selected pool. The cells were then expanded and subsequently confirmed by pluripotency assays.

[0202] g. Induction and identification of iPSC-derived endothelial cells (iECs) iECs were induced using a three-dimensional approach. Briefly, to initiate differentiation, leukemia inhibitory factor (LIF)-dependent iPSCs were cultured in ultra-low, non-adherent dishes in EBM2 medium (Lonza) to generate embryoid bodies (EBs). After 4 days of suspension culture, the EBs were plated on a 0.2% gelatin-coated The cells were allowed to reattach to the dish and then incubated with VEGF-A165 (50 ng / mL; Peprotech). After 3 weeks of differentiation, the cells were cultured in EBM2 medium supplemented with 0.1% DMSO. Single cell suspensions were obtained using a cytometer (Life Technologies) and then incubated with APC-bound CD31 (eBi iECs were labeled with anti-mouse antibodies against CD144 (BD Biosciences) and PE-conjugated CD144 (BD Biosciences). The CD31+ CD144+ population was analyzed by fluorescence activated cell sorting. iECs were purified by FACS. The cells were maintained in supplemented EBM2 medium.

[0203] Their phenotype was characterized by immunofluorescence for CD31 and VE-cadherin, as well as PCR and tubule morphology. In a maturation assay (an assay to demonstrate endothelial function in forming premature vascular structures), Note: Confluent iPSC monolayers on 0.1% gelatin or Matrigel were cultured. The differentiation protocol using this medium was also successful. Note: Other endothelial cell media have also been used successfully.

[0204] h. Induction and identification of iPSC-derived smooth muscle cells (iSMCs): Resuspended iPSCs were cultured in 6-well plates. , 0.1% gelatin-coated plastic Petri dishes (Falcon, Becton-Dicken 20 cells / well were cultured in 2 ml of differentiation medium containing 10 μM of 10-HT 2-HT 200 mM NaCl at 37°C and 5% CO2. The differentiation medium was prepared by mixing DMEM, 15% fetal bovine 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. .

[0205] From day 11, the differentiation medium was changed to serum-free medium of knockout DMEM: 15% knockout serum. Substitutes, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin, and streptavidin. The cultures were maintained for an additional 10 days with daily changes of serum-free medium. The phenotype was characterized by immunofluorescence and PCR for both SMA and SM22. Confirmed.

[0206] i. Induction and identification of iPSC-derived cardiomyocytes (iCMs) Prior to differentiation, iPSCs were cultured on gelatin-coated The feeder cells were removed by passage twice on a culture dish containing iPSCs. The cells were dissociated with TrypLE (Invitrogen) and incubated at 75,000 x g for 1 h without the addition of additional growth factors. Three-day-old EBs were dissociated and the cells were cultured at -100,000 cells / ml for 48 hours. Briefly, 6 × 10 4 - 10×10 4 Cells were incubated with 2 mM L-glutamate MIN, 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). StemPro-34 SF medium (Invitrogen) was used to culture the cells in gelatin-coated 96-well flat-bottom plates. (Becton Dickinson, Franklin Lakes, NJ) Wells were seeded with 0.1% ethanol and 0.1% ethanol. Cultures were harvested after 4 or 5 days (total of 7 or 8 days).

[0207] Their phenotype was characterized by the expression of troponin I and sarcomeric alpha actinin. The results were confirmed by IF for α-Gata4 and α-Mhy6. The cells began to beat within 8–10 days, demonstrating their functional differentiation. .

[0208] j. Induction and identification of iPSC-derived islet cells (iICs) iPS cells were cultured on gelatin-coated plates. The feeder layer was removed by transferring the cells to a sco Non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, and 1 μg / mL rhamnosine 1 × 10 cells / well in DMEM / F-12 medium with 10% FBS supplemented with 5% ribosomal protein. 6 Cell Colla ES-D3 cells were seeded on gen I-coated plates and left overnight. 100 μM non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, 25 μg / ml The cells were cultured in DMEM / F-12 medium with 2% FBS supplemented with 6 mL insulin and 1 μg / mL laminin. The phenotypes of these mice were characterized by immunofluorescence for c-peptide, glucagon, Ngn3, and Amylase, insulin 2, somatostatin and insulin production were detected by PCR. I confirmed this.

[0209] k. Induction and identification of iPSC-derived neural cells (iNCs) To initiate the monolayer protocol, iPSCs were cultured in vitro. The cells were gently dissociated and then placed in RHB-A or N2B27 medium (StemCell Science Inc.). , 1×10 4 cells / cm 2 Plate on 0.1% gelatin-coated tissue culture plastic at 1 °C for 1 h. The medium was changed every other day. For reseeding on day 4, cells were dissociated and resuspended in 5 ng / ml mouse Laminin-coated cells were grown in RHB-A medium supplemented with bFGF (Peprotech). On tissue culture plastic, 2 x 10 4 cells / cm 2 From this point on, cells were plated every 4 days. The cells were then reseeded under the same conditions every 2 days and cultured for a total of 20 days. To quantify the number of differentiated neurons, cells were plated in laminar cultures in 24-well Nunc plates. Two days after seeding, the medium was replaced with RHB-A:Neu The robasal:B27 mixture (1:1:0.02) was used to improve the survival of differentiated neurons. Their phenotypes were confirmed by IF for Tuj-1 and nestin.

[0210] l. Elispot assay Unidirectional enzyme-linked immunospot (ELISA) assay For the Enzyme-Linked ImmunoSpot (Elispot) assay, cells (miPSCs, mi PSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m- / - Ciita- / - Cd47 tg) Five days after the injection, recipient splenocytes were isolated from fresh spleens and used as responder cells. Donor cells (miPSC, miPSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m- / - Ciita- / - Cd47 tg) were inhibited by mitomycin and served as stimulator cells. 6 5 × 10 stimulator cells 5 Incubated with recipient responder splenocytes for 24 hours and then incubated with I The spot frequencies of FNγ and IL-4 were automatically measured using an ELISPOT plate reader. All assays were performed in quadruplicate.

[0211] m. In vivo Teratoma assay to study iPSC survival in 6-week-old syngeneic or allogeneic cultured The mice were used for transplantation of wtiPSCs or non-immunogenic iPSCs cells. 6 1 cell 00 μl was injected into the right thigh muscle of the mice. The transplanted animals were observed regularly every other day. Tumor growth was measured with a caliper. 3 After the tumor has grown to more than 100 After a 3-day observation period, they were sacrificed.

[0212] n. In vitro NK cell assay in vitro NK cells after co-culture with wt iPSCs or HIP cells CD107 expression on NK cells was measured by flow cytometry as a marker of NK cell activation. Using the ELISPOT principle, NK cells were co-cultured with wt iPSCs or HIP cells, and NK The release of IFN-γ from the cells was measured.

[0213] According to the "missing self theory," MHC I-deficient stem cells can be expressed in mice and Both human and human PSCs express ligands that activate NK receptors, and thus NK It has been demonstrated that the expression of this activating receptor decreases with differentiation. Although it has been reported that NK killing of B2K- / - derivatives is reduced, HLA-E and HLA-G are expressed separately in human pluripotent stem cells. This has been used in the hope that innate immune responses in HLA I − / − cells may be reduced. However, among them there are additional inhibitory non-MHC ligands that are highly effective. These results reveal that Cd47 is a surprisingly potent inhibitor of innate immune clearance.

[0214] o. Summary of mouse data All engineered miPSC lines were cultured without immunosuppression. , and transplanted into syngeneic C57BL / 6 and allogeneic BALB / c recipients. The ES cells grew into teratomas in the syngeneic recipients, but not in the allogeneic recipients. In mice, the survival of the modified cells depended on their level of low immunogenicity. Teratoma formation by miPSCs was 60% in BALB / c mice, with little ELISPOT response and However, measurable IgM antibody responses were observed. B2m- / - Ciita- / - miPSCs were Teratoma formation was observed in 91.7% of ALB / c mice, and slight ELISPOT reaction was observed, but no antibody reaction was observed. The final B2m- / - Ciita- / - Cd47 tg miPSC line showed 100% teratoma formation. , and did not show ELISPOT or antibody responses. We performed an assay to confirm the association between B2m- / - Ciita- / - miPSCs and B2m- / - Ciita- / - Cd47 tg miPSCs. This was further evaluated by comparing the CD47 overexpression with the CD107 Expression of IFN-γ and release of IFN-γ from NK cells were significantly decreased, resulting in milder innate immune clearance. In summary, with each modification step, the miPSCs became less immunogenic. I did.

[0215] B2m- / - Ciita- / - HIP cells generate low immunogenic endothelial-like cells (miECs) and smooth muscle-like cells (miSMCs). ), and cardiomyocyte-like cells (miCMs). s) (i.e., derived from unmodified miPSCs) were used as controls. derivatives) are intended to be derived from the typical morphological appearance, cell Immunofluorescence and gene expression of markers are shown. MHC I in wt derivatives The expression of II and II molecules was generally significantly upregulated compared to their parental miPSC lines. As expected, miECs expressed MHC I and MHC II. expression of MHC I and II was by far the highest in the ES cells, whereas miSMCs had intermediate expression of MHC I and II. In contrast, miCMs expressed moderate levels of MHC I but very low levels of MHC II. The derivatives showed significantly lower expression of Cd47, but slightly higher than miPSCs. All B2m- / - Ciita- / - Cd47 tg derivatives expressed MHC I and Complete lack of MHC II, as well as significantly higher Cd47 than their wt counterparts, showed.

[0216] 5×10 5 Matrigel plugs containing wt miECs, miSMCs, and miCMs were prepared syngeneically. The cells were then implanted into the subcutaneous plexus of C57BL / 6 or allogeneic BALB / c mice. After 5 days, all allogeneic Recipients mount strong cellular immune responses and In sharp contrast, the corresponding B2m- / - Ciita- / - mice mounted strong IgM antibody responses. None of the Cd47 tg(HIP) derivatives increased IFN-γ ELISPOT frequency or No increase in IgM antibody production was detected.

[0217] The morphology of the transplanted cells was also confirmed. Allogeneic mice were placed in an induction chamber and incubated with 2% isoflurane. Anesthesia was induced with 250 μl of 100 mM NaCl in 0.9% saline (Isothesia, Butler Schein). 1 million (1 mio) cells (HIP miPSC-derived cardiomyocytes (miCMs), HIP miPSC-derived smooth muscle cells ( miSMCs or HIP miPSC-derived endothelial cells (miECs) were cultured in 250 μl of BD Matrigel High Concentration Solution. The mixture was mixed with 1:1 BD Biosciences solution and injected into the lower back of the mouse using a 23-G syringe. The Matrigel plugs were injected subcutaneously at 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks after implantation. The tissue was then fixed in 4% paraformaldehyde and 1% glutenaldehyde for 24 hours. They were then dehydrated and embedded in paraffin. 5 μm thick sections were cut and analyzed by hematoxylin-eosin. Histological examination revealed morphologically consistent miCMs and miCMs. S.M. Cs, and miECs were confirmed.

[0218] G. Example 5: Generation of human iPSCs Human episomal iPSC lines were generated using three plasmids , seven factors (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen), using Thermo Fisher's EBNA-based episomal system, CD34+ umbilical cord blood cells were Derived from cord blood (catalog no. A33124, Thermo Fisher Scientific) This iPSC line will allow us to determine what is integrated into the genome during the reprogramming event. It is considered to have a zero footprint because it has no It has also been shown that the iPSCs do not contain reprogramming genes. Karyotype and OCT4, SOX2, NANOG (as shown by RT-PCR), OCT4, SSEA 4, pluripotency markers such as TRA-1-60 and TRA-1-81 (as shown by ICC) These hiPSCs express endogenously in directed differentiation and teratoma analysis. The cells retained the differentiation potential into ectodermal, endodermal, and mesodermal lineages. and cardiac lineages were induced with extremely stable efficiency.

[0219] Note: Several gene delivery vehicles have been successfully used to generate iPSCs. These include retroviral vectors, adenoviral vectors, Sendai virus, and virus-free reprogramming methods (episomal vectors, piggyBac vectors, etc.) Uses include amplicons, synthetic mRNAs, microRNAs, recombinant proteins, and small molecule drugs. (These include

[0220] Note: Also known as the Yamanaka factor, OCT3 / 4 , SOX2 , KLF4 , and C-MYC First reported in Various factors, including combinations of different factors, could be successfully used for reprogramming. In an embodiment, only three of these factors are combined, and C-MYC Successfully excluded However, the reprogramming efficiency was reduced.

[0221] In one embodiment, C-MYC instead of L-MYC or GLIS1 This improves reprogramming efficiency. In another embodiment, the reprogramming factors are not limited to genes associated with pluripotency. Not determined.

[0222] Statistics All data are presented as mean ± SD or as median and minimum-maximum range. Differences between groups were evaluated using unpaired Student's mean mean ± SD. The results were analyzed using either a one-way t-test or a one-way analysis of variance (ANOVA) with Bonferroni post hoc test. *p<0.05, **p<0.01.

[0223] H. Example 6: Generation of human HIP cells Human Cas9 iPSCs undergo two gene editing steps. In the first step, the CRISPR technique was performed, and the CRISPR sequence 5'-CGTGAGTAAACCTGA ATCTT-3' for the coding sequence of the human β-2-microglobulin (B2M) gene, and CRIS The coding sequence of the human CIITA gene was inserted into the PR sequence 5'-GATATTGGCATAAGCCTCCC-3'. The kit instructions (MEGAshortscript T7 Transcription Kit, Thermo According to the method described by Fisher et al., a linearized CRISPR sequence with a T7 promoter was used to generate gRNA. was synthesized. In vitro Transcribed (IVT) gRNA was analyzed using MEGAclear Transcription Clean For IVT gRNA delivery, 300 ng IV-Up Kit was used to deliver the gRNA to single cells. T gRNA was electroporated using the Neon electroporation system. After electroporation, the edited Cas9 iPSCs were plated as single cells and expanded. iPSC cultures were dissociated into single cells using TrypLE (Gibco). Tra1-60 Alexa Fluor (登録商標) Stained with 488 and propidium iodide (PI) Cell sorting was performed using a FACS Aria cell sorter (BD Biosciences) using forward scattering and By selectively gating on the side scatter emission and the side scatter emission, the two clumps and debris were isolated from the seeded cells. Viable pluripotent cells were identified as cells that were not stained with PI and stained with Tra1-60 Alexa Fluor 3 (Alexa Fluor 3) and were excluded from the study. The single cells were then selected for staining with Fluor 488. The cells were then grown until they reached a population size of 100, and the colonies were then tested for CRISPR editing. Cleavage was detected using the GeneArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific). Genomic DNA was assessed using 1 × 10 6 hiPSCs were isolated from 1000 cells and purified using AmpliTaq Gold 360. For Master Mix and B2M, F: 5'-TGGGGCCAAATCATGTAGACTC -3' and R: 5'- TCAGTGGGGGTGAATTCAGTGT-3', and for CIITA F: 5'-CTTAACAGCGATGCTGACCCC A: 5'-TGGCCTCCATCTCCCCTCTCTT-3' and B: 5'-TGGCCTCCATCTCCCCTCTT-3' were used to amplify B2M. Genomic DNA regions of CIITA and CIITA were amplified by PCR. Clean up the material (PureLink PCR Purification Kit, Thermo Fisher Scientific) Sanger sequencing was performed to predict indel frequency. After confirming that the samples were out- sourced, karyotype analysis and TaqMan hPSC Scorecard Panel (Ser) were performed. The cells were further characterized by immunofluorescence (IHC) and immunofluorescence staining (IHC-Fisher) performed. and maintained a normal (46,XX) karyotype during the genome editing process.

[0224] In the second step, the CD47 gene was synthesized and its DNA was inserted into the EF1a promoter and p53 promoter. A plasmid carrying romycin resistance was cloned into the lentivirus. 1 x 10 7 TU / mL lentivirus stock and 6 μg / mL polybrene (Thermo Fisher Scientific) were used. After transduction, the medium was changed every day. Three days after transduction, the cells were Cells were grown and selected with 0.5 μg / mL puromycin. After selection with 100 μg / ml of 10 ... The levels of CD47 were confirmed by qPCR. To this end, we used pluripotency assays (TaqMan hPSC Scorecard Panel, Thermo Fisher Scientific) and nuclear The typing analysis was repeated.

[0225] I. Example 7: Differentiation of human HIP cells 1. Differentiation of hHIP cells into human cardiomyocytes This is described in Sharma et al., J. Vis Exp. 2015 doi: 10.3791 / 52628 (in its entirety and in particular in cells) The techniques for differentiation are adapted from those described in the above, which are incorporated herein by reference. The procedure was performed using the protocol described in the previous section. hiPSCs were cultured in diluted Matrigel medium in 6-well plates. The cells were seeded on a 100% PBS (356231, Corning Medical) and cultured in Essential 8 Flex medium (Thermo Fisher Scientific). When the cells reached 90% confluency, they were differentiated. The medium was replaced with insulin-free 2% B-27 (Gibco) and 6 μM CHIR-9902. The medium was replaced with 5 mL of RPMI 1640 (Gibco) containing 1 (Selleck Chem). After 3 days, the medium was replaced with RPMI1640 containing 2% B-27 without insulin and without CHIR. On the fifth day, 5 μL of IWR1 was added to the medium and cultured for another 2 days. The cells were then transferred back to RPMI1640 medium containing 2% B-27 without insulin and incubated for 48 hours. On the day, the medium was replaced with RPMI1640 containing insulin-containing B27 (GIBCO), and then The medium was replaced with the same medium every 3 days after the incubation. Spontaneous beating of cardiomyocytes began to occur approximately from day 10 to day 12. On the 10th day after differentiation, cardiomyocytes were purified. Briefly, the medium was replaced with low glucose medium and maintained for 3 days. On day 13, The medium was changed back to RPMI1640 containing insulin-containing B27. This procedure was repeated on day 14. The remaining cells are highly purified cardiomyocytes.

[0226] 2. Differentiation of hHIP cells into human endothelial cells. hiPSCs were cultured in diluted PBS in a 6-well plate. The cells were seeded on Trigel (356231, Corning) and cultured in Essential 8 Flex medium (ThermoFisher). When the cells reached 60% confluency, Differentiation was initiated and the medium was changed to insulin-free 2% B-27 (Gibco) and 5 μM CHI. The medium was replaced with RPMI 1640 (Gibco) containing R-99021 (Selleck Chem). On the day of incubation, the medium was reduced to: 2% B-27 (GIBCO) without insulin and 2 μM CHIR- The medium was then changed to RPMI 1640 (Gibco) containing 99021 (Selleck Chem). From days 4 to 7, cells were cultured in RPMI EC medium, i.e., insulin-free 2% B-27, 50 ng / ml Vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 10 ng / mL ml fibroblast growth factor basic (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich) Dowitch, St. Louis, MO, USA) and 1 μM SB 431542 (Sigma-Aldrich) Endothelial cell clusters were visible from day 7 onwards. The cells were cultured in 10% FCS hi (Gibco), 25 ng / ml vascular endothelial growth factor (VEGF; R&D System) and 10% erythrocyte sedimentation buffer (100 mL). tem, Minneapolis, MN, USA), 2 ng / ml fibroblast growth factor basic (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich, St. Louis, MO) EGM-2 SingleQu (Emerald City, CA, USA) and 1 μM SB 431542 (Sigma-Aldrich) The differentiation process was completed after 14 days. During the differentiation process, undifferentiated cells were detached. For purification, the cells were purified by expressing CD31 Robbie's (Miltenyi, Auburn, CA) The highly purified IgG was processed for MACS according to the manufacturer's protocol. EC cells were cultured in EGM-2 SingleQuots medium supplemented with supplements and 10% FCS hi (GIBCO). The cells were cultured in 100% ethanol (Lonza, Basel, Switzerland) and incubated for 3 to 4 days using TrypLE. Cells were passaged 1:3.

[0227] J. Transplantation into Humanized Mice Humanized NSG-SGM3 mice were placed in an induction chamber and incubated with 2% isopropanol. Anesthesia was induced with flurane (Isothesia, Butler Schein). Benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone, Carby Ochem), Bcl-XL BH 4 (cell-permeable TAT peptide, 50 nM, Calbiochem), Cyclosporine Porin A (200 nM, Sigma), IGF-1 (100 ng / ml, Peprotec), and pinacidil (50 μM One million (1 mio) cells (hiPSC-derived cardiomyocytes) were cultured in 0.9% saline containing 100 mM NaCl (Sigma). hiPSC-derived endothelial cells (hiCMs) or hiPSC-derived endothelial cells (hiECs) were cultured in 250 μl of BD Matrigel. The mixture was mixed with high-concentration EDTA (1:1; BD Biosciences) and then injected into the 23-G syringe. The mice were subcutaneously injected into the lower back using the same method. Matrigel showed a 2, 4, 6, 8, 10, and 12 week post-implantation The plugs were then excised and fixed in 4% paraformaldehyde and 1% glutenaldehyde for 24 hours. The tissue was then fixed for 1 h at 4 °C for 1 h, then dehydrated and embedded in paraffin. Sections of 5 μm thickness were cut and The morphology was confirmed by staining with matoxylin and eosin (HE).

[0228] IX. Exemplary Sequences: SEQ ID NO:1 - Human beta-2-microglobulin MSRSVALAVLALLSLSG LEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDE YACRVNHVTLSQPKIVKWDRDI

[0229] SEQ ID NO:2 - Human CIITA protein, 160 amino acids N-term MRCLAPRPAGSYLSEPQGSSQCATME LGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVF QDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKP

[0230] SEQ ID NO:3 - human CD47MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKW KFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFS PNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTG ILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE

[0231] SEQ ID NO:4 - Herpes simplex virus thymidine kinase (HSV-tk) MASYPCHQHASAFDQAA RSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETI ANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPA ARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGS WWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPA GCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN

[0232] SEQ ID NO:5 - Escherichia coli ( Escherichia coli )Cytosine deaminase (EC-CD)MSNNALQTIIN ARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAE RKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLE EALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSRFVETVAALAHHEGMGARVTASHTTAM HSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQV LHMGLHVCQLMGYGQINDGLNLITHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIASTQPA QTTVYLEQPEAIDYKR

[0233] SEQ ID NO:6 - truncated human caspase 9GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCR ESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAIS SLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFF KTS

[0234] All publications and patent documents disclosed or referenced herein are incorporated by reference in their entirety. The foregoing description has been presented for purposes of illustration and description only. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. It is intended that the scope of the present invention be defined by the claims appended hereto.< / nu> < / nu>

Claims

1. A method for producing low immunogenic pluripotent stem cells, comprising: a. induced pluripotent stem cells (iPS C) abolishing the activity of both alleles of the B2M gene in said iPSCs; abolishing the activity of both alleles of the CIITA gene in said iPSCs; and c. Increased expression of CD47 in erythrocytes.

2. The iPSCs are human, the B2M gene is human, and the CIITA gene is human. and the increased expression of CD47 is due to the expression of the human CD47 gene under the control of the promoter. The method of claim 1, resulting from introducing at least one copy of the gene into said iPSC cell. 。

3. The iPSC is mouse, the B2m gene is mouse, and the Ciita gene is mouse. and the increased expression of Cd47 is under the control of a mouse C The method of claim 1, resulting from the introduction of at least one copy of the d47 gene into said iPSC cell. Method 1.

4. The method according to claim 2 or 3, wherein the promoter is a constitutive promoter. Law.

5. The disruption in both alleles of the B2M gene is Clustered, regularly spaced, short palindromic repeats that disrupt both Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 (CRISPR 4. The method according to claim 1 , wherein the reaction is carried out by the reaction:

6. The disruption in both alleles of the CIITA gene is The method according to any one of claims 1 to 3, wherein the method is performed by a CRISPR reaction that disrupts both genes. 。

7. Human hypoimmunogenic pluripotent (hHIP) stem cells containing: a. both copies of the endogenous B2M gene one or more modifications that inactivate an allele; b. both alleles of the endogenous CIITA gene one or more modifications that inactivate a gene; and c. one or more modifications that inactivate a CD47 gene in said hHIP stem cells. wherein said hHIP stem cells have the B2M and CIITA modifications. The results showed that the CD47 gene expression was increased by induced pluripotent stem cells (iPSCs) containing the CD47 gene but not the CD47 gene. The primary natural killer (NK) cell response was lower than the secondary NK cell response induced by the and wherein said first and second NK cell responses are generated by said hHIP cells or said In vitro incubation with either of the iPSC cells containing only the B2M and CIITA modifications The effect of IFN-γ on the immune response is measured by determining the levels of IFN-γ from immunized NK cells.

8. Human hypoimmunogenic pluripotent (hHIP) stem cells containing: a. both copies of the endogenous B2M gene one or more modifications that inactivate an allele; b. both alleles of the endogenous CIITA gene one or more modifications that inactivate a gene; and c. one or more modifications that inactivate a CD47 gene in said hHIP stem cells. wherein said hHIP stem cells are modified to increase expression of the same gene(s) induced by iPSCs; lower secondary T cell responses in allogeneic humanized mouse strains and inducing a first T cell response in said first and second T cells. The response was measured by measuring IFN-γ levels from splenocytes of the humanized mice in an ELISPOT assay. It is measured by determining

9. A method comprising transplanting the hHIP stem cells of any one of claims 7 or 8 into a human subject. 。

10. Low immunogenic pluripotent cells, comprising: a. a reduced endogenous pluripotent cell when compared to the parent pluripotent cell intrinsic major histocompatibility antigen class I (HLA-I) function when compared to the parental pluripotent cells; c. decreased endogenous major histocompatibility antigen class II (HLA-II) function; and augmentation of CD47 function that reduces susceptibility to killing by the low immunogenic pluripotent stem cells; The cells exhibited reduced HLA-I function, reduced HLA-II function, and reduced ability to be killed by NK cells. As a result of their decreased sensitivity to erythrocytes, they are less susceptible to rejection when transplanted into a subject.

11. The HLA-I function is mediated by downregulating the expression of the β-2 microglobulin protein. The method of claim 10, wherein the immunogenicity of the pluripotent cell is reduced by the administration of the low immunogenic pluripotent cell.

12. The gene encoding the beta-2 microglobulin protein has been knocked out The low immunogenic pluripotent cell of claim 11.

13. The beta-2 microglobulin protein has a sequence identity of at least 90% to SEQ ID NO:

1. The low immunogenic pluripotent cell of claim 12, having an identity.

14. 14. The method of claim 13, wherein the beta-2 microglobulin protein has the sequence of SEQ ID NO:

1. A hypoimmunogenic pluripotent cell as described herein.

15. The HLA-I function is reduced by reducing expression of HLA-A protein. The low immunogenic pluripotent cell according to item 10.

16. The method according to claim 15, wherein the gene encoding the HLA-A protein is knocked out. Low immunogenic pluripotent cells.

17. The HLA-I function is reduced by reducing expression of HLA-B protein. The low immunogenic pluripotent cell according to item 10.

18. The hypoimmunogenic pluripotent cell of claim 17, wherein the HLA-B protein is knocked out. 。

19. The HLA-I function is reduced by reducing expression of HLA-C protein. Item 11. The low immunogenic pluripotent cell according to item 10.

20. The method according to claim 19, wherein the gene encoding the HLA-C protein is knocked out. Low immunogenic pluripotent cells.

21. 21. The method of claim 10, wherein the hypoimmunogenic pluripotent cells do not contain HLA-I function. A low immunogenic pluripotent cell according to claim 1.

22. The HLA-II function is reduced by reducing the expression of the CIITA protein.

22. The low immunogenic pluripotent cell according to any one of items 10 to 21.

23. The method according to claim 22, wherein the gene encoding the CIITA protein is knocked out. Low immunogenic pluripotent cells.

24. The CIITA protein has at least 90% sequence identity to SEQ ID NO:2; The low immunogenic pluripotent cell of claim 23.

25. The low immunogenicity of claim 24, wherein the CIITA protein has the sequence of SEQ ID NO:

2. Pluripotent cells.

26. The HLA-II function is reduced by reducing the expression of HLA-DP protein.

22. The low immunogenic pluripotent cell of any one of claims 10 to 21.

27. The method according to claim 26, wherein the gene encoding the HLA-DP protein is knocked out. Low immunogenic pluripotent cells.

28. The HLA-II function is reduced by reducing the expression of HLA-DR protein.

22. The low immunogenic pluripotent cell of any one of claims 10 to 21.

29. 29. The method of claim 28, wherein the gene encoding the HLA-DR protein is knocked out. Low immunogenic pluripotent cells.

30. The HLA-II function is reduced by reducing the expression of HLA-DQ protein.

22. The low immunogenic pluripotent cell of any one of claims 10 to 21.

31. The method according to claim 30, wherein the gene encoding the HLA-DQ protein is knocked out. Low immunogenic pluripotent cells.

32. 32. The method of claim 10, wherein the hypoimmunogenic pluripotent cells do not contain HLA-II function. A low immunogenic pluripotent cell according to claim 1.

33. The decreased susceptibility to killing by NK cells was due to increased expression of the CD47 protein. The poorly immunogenic pluripotent cell of any one of claims 10 to 32, which is caused by

34. The increased expression of the CD47 protein results from an alteration to an endogenous CD47 locus.

34. The low immunogenic pluripotent cell according to claim 33.

35. 34. The method of claim 33, wherein the increased expression of CD47 protein results from a CD47 transgene. of low immunogenic pluripotent cells.

36. the CD47 protein has at least 90% sequence identity to SEQ ID NO:3; 36. The low immunogenic pluripotent cell of any one of claims 33 to 35.

37. 28. The low immunogenic polypeptide of claim 27, wherein the CD47 protein has the sequence of SEQ ID NO:

3. Competent cells.

38. The method further comprises the step of: detecting a suicide gene that is activated by a trigger that causes the hypoimmunogenic pluripotent cells to die. The low immunogenic pluripotent cell of any one of claims 10 to 37.

39. the suicide gene is the herpes simplex virus thymidine kinase gene (HSV-tk); The hypoimmunogenic pluripotent cell of claim 38 , wherein the trigger is ganciclovir.

40. The HSV-tk gene is a protein that contains at least 90% sequence identity to SEQ ID NO:

4. The hypoimmunogenic pluripotent cell of claim 39 , encoding a protein.

41. 41. The method of claim 40, wherein the HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:

4. A hypoimmunogenic pluripotent cell as described herein.

42. The suicide gene is the Escherichia coli cytosine deaminase gene (EC-CD), and the trigger is The low immunogenic pluripotent cell of claim 38, which is 5-fluorocytosine (5-FC).

43. The EC-CD gene is a protein having at least 90% sequence identity to SEQ ID NO:

5. The hypoimmunogenic pluripotent cell of claim 42, encoding a gene encoding a pluripotent protein.

44. 44. The method of claim 43, wherein the EC-CD gene encodes a protein comprising the sequence of SEQ ID NO:

5. A hypoimmunogenic pluripotent cell as described herein.

45. The suicide gene encodes an inducible caspase protein and the trigger is a protein that induces dimerization. The hypoimmunogenic pluripotent cell of claim 38, which is a chemical inducer (CID).

46. The gene is an inducible caspase gene that contains at least 90% sequence identity to SEQ ID NO:

6. The hypoimmunogenic pluripotent cell of claim 45, which encodes a β-K+ protein.

47. The gene encodes an inducible caspase protein comprising the sequence of SEQ ID NO:

6.

47. The low immunogenic pluripotent cell of claim 46.

48. 48. The low immunogenic multipotency antibody of any one of claims 45 to 47, wherein the CID is AP1903. sex cells.

49. A method for producing a low immunogenic pluripotent cell comprising: a. an endogenous major gene present in the pluripotent cell; a. Reduce histocompatibility antigen class I (HLA-I) function; b. Intrinsic factors present in pluripotent cells Sexual major histocompatibility antigen (MHA) clade and c. reducing human leukocyte antigen II (HLA-II) function; and It increases the expression of proteins that decrease the sensitivity of cells.

50. The HLA-I function is mediated by downregulating the expression of the β-2 microglobulin protein.

50. The method of claim 49, wherein the concentration of

51. The expression of the beta-2 microglobulin protein is The protein of claim 50 is reduced by knocking out a gene encoding the protein. method.

52. The beta-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO:

1. β-2 microglobulin 50.

53. The beta-2 microglobulin protein has the sequence of SEQ ID NO:

1. Roblin 51.

54. The HLA-I function is decreased by decreasing the expression of HLA-A protein expression.

50. The method of claim 49.

55. The expression of the HLA-A protein is caused by knocking out the gene encoding the HLA-A protein.

55. The method of claim 54, wherein the concentration is reduced by reducing the concentration of

56. The HLA-I function is decreased by decreasing the expression of HLA-B protein expression.

50. The method of claim 49.

57. The expression of the HLA-B protein is achieved by knocking out the gene encoding the HLA-B protein.

57. The method of claim 56, wherein the amount of the compound is reduced by increasing the amount of the compound.

58. The HLA-I function is decreased by decreasing the expression of HLA-C protein expression.

50. The method of claim 49.

59. The expression of the HLA-C protein is achieved by knocking out the gene encoding the HLA-C protein.

59. The method of claim 58, wherein the method is further characterized by:

60. 60. Any one of claims 49 to 59, wherein the hypoimmunogenic pluripotent cells do not contain HLA-I function. The method described above.

61. The HLA-II function is reduced by reducing the expression of the CIITA protein.

61. The method of any one of claims 49 to 60.

62. The expression of the CIITA protein knocks out the gene encoding the CIITA protein.

61. The method of claim 60, wherein the concentration is reduced by lowering the concentration.

63. The CIITA protein has at least 90% sequence identity to SEQ ID NO:2; 62. The method of claim 61.

64. The method of claim 63, wherein the CIITA protein has the sequence of SEQ ID NO:

2.

65. The HLA-II function is reduced by reducing the expression of HLA-DP protein.

61. The method of any one of claims 49 to 60.

66. Expression of the HLA-DP protein is achieved by knocking out the gene encoding the HLA-DP protein.

66. The method of claim 65, wherein the decrease is caused by out.

67. The HLA-II function is reduced by reducing the expression of HLA-DR protein.

61. The method of any one of claims 49 to 60.

68. Expression of the HLA-DR protein is achieved by knocking out the gene encoding the HLA-DR protein.

68. The method of claim 67, wherein the decrease is caused by out.

69. The HLA-II function is reduced by reducing the expression of HLA-DQ protein.

61. The method of any one of claims 49 to 60.

70. Expression of the HLA-DQ protein is achieved by knocking out the gene encoding the HLA-DQ protein.

70. The method of claim 69, wherein the decrease is caused by out.

71. 71. Any one of claims 49 to 70, wherein the hypoimmunogenic pluripotent cells do not contain HLA-II function. The method described above.

72. A protein that reduces the susceptibility of said pluripotent cells to phagocytosis by macrophages.

72. The method of claim 49, wherein the increased expression of the gene is caused by an alteration to an endogenous gene locus.

3. The method according to claim 1 .

73. The method of claim 72, wherein the endogenous locus encodes a CD47 protein.

74. Any of claims 49 to 71, wherein the increased expression of the protein occurs by expression of a transgene. The method according to any one of claims 1 to 5.

75. 75. The method of claim 74, wherein the transgene encodes a CD47 protein.

76. the CD47 protein has at least 90% sequence identity to SEQ ID NO:3; 75. A method according to any one of claims 73 or 74.

77. The method of claim 76, wherein the CD47 protein has the sequence of SEQ ID NO:

3.

78. A suicide gene is expressed that is activated by a trigger that kills the low immunogenic pluripotent cells.

78. The method of any one of claims 49 to 77, further comprising:

79. the suicide gene is the herpes simplex virus thymidine kinase gene (HSV-tk); 79. The method of claim 78, wherein the trigger is ganciclovir.

80. The HSV-tk gene is a protein that contains at least 90% sequence identity to SEQ ID NO:

4.

80. The method of claim 79, further comprising encoding a protein.

81. 81. The method of claim 80, wherein the HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:

4. method.

82. The suicide gene is the Escherichia coli cytosine deaminase gene (EC-CD), and the trigger is The method of claim 78, wherein the amino acid is 5-fluorocytosine (5-FC).

83. The EC-CD gene is a protein having at least 90% sequence identity to SEQ ID NO:

5.

83. The method of claim 82, wherein the quality is coded.

84. 84. The method of claim 83, wherein the EC-CD gene encodes a protein comprising the sequence of SEQ ID NO:

5. The method described.

85. The suicide gene encodes an inducible caspase protein and the trigger is a protein that induces dimerization.

79. The method of claim 78, which is a specific chemical inducer (CID).

86. The gene is an inducible caspase gene that contains at least 90% sequence identity to SEQ ID NO:

6. The method of claim 85, wherein the gene encodes a ze protein.

87. The gene encodes an inducible caspase protein comprising the sequence of SEQ ID NO:

6.

87. The method of claim 86.

88. 88. The method of any one of claims 85 to 87, wherein the CID is AP1903.

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