method for generating a hypoimmunogenic pluripotent stem cell, human hypoimmunogenic pluripotent ste

专利摘要:
the invention provides pluripotent cells that are used therapeutically to regenerate tissues, but prevent rejection by individuals who receive them. in particular, the invention provides hypoimmunogenic pluripotent cells that prevent host immune rejection. the cells lack important immune antigens that trigger immune responses and are designed to prevent phagocytic endocytosis. the invention also provides universally acceptable pluripotent shelf cells and derivatives thereof to generate or regenerate specific tissues and organs.
公开号:BR112019014257A2
申请号:R112019014257
申请日:2018-01-14
公开日:2020-04-28
发明作者:SCHREPFER Sonja；Deuse Tobias
申请人:Univ California；
IPC主号:

专利说明:

METHOD FOR GENERATING A HYPHOIMMUNOGENIC PLURIPOTENT STEM CELL, HUMAN HYPHOIMMUNOGENIC PLURIPOTENT STEM CELL, METHOD FOR PRODUCTION OF A HYPOIMMUNOGENIC PLURIPOTENT CELL, AND MICROGLOBULINE β-2
I. CROSS REFERENCE TO RELATED APPLICATIONS [001] This application claims the benefit of U.S. Provisional Application No. 62 / 445,969, filed on January 13, 2017.
II. FIELD OF THE INVENTION [002] Regenerative cell therapy is an important potential treatment for the regeneration of injured organs and tissues. With the low availability of organs for transplantation and the long wait that accompanies it, the possibility of tissue regeneration by transplanting cell lines readily available in patients is understandably attractive. Regenerative cell therapy has shown promising initial results for the rehabilitation of damaged tissues after transplantation in animal models (for example, after myocardial infarction). The propensity for the transplant recipient's immune system to reject allogeneic material, however, greatly reduces the potential efficacy of the therapy and lessens the possible positive effects around such treatments.
III. BACKGROUND OF THE INVENTION [003] Regenerative cell therapy is an important potential treatment for the regeneration of injured organs and tissues. With the low availability of organs for transplantation and the long wait that accompanies it, the possibility of tissue regeneration by transplanting cell lines readily available in patients is
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2/105 understandably attractive. Regenerative cell therapy has shown promising initial results for the rehabilitation of damaged tissues after transplantation in animal models (for example, after myocardial infarction). The propensity for the transplant recipient's immune system to reject allogeneic material, however, greatly reduces the potential efficacy of the therapy and lessens the possible positive effects around such treatments.
[004] Autologous induced pluripotent stem cells (iPSCs) are theoretically an unlimited cell source for patient-specific organ-based organ repair strategies. Its generation, however, presents technical and manufacturing challenges and is a lengthy process that conceptually prevents any type of acute treatment. Allogeneic therapies based on iPSC are easier from a manufacturing point of view and allow the generation of standardized and high quality cellular products. Due to their allogeneic origin, however, such cellular products would be rejected. With the reduction or elimination of cell antigenicity, universally acceptable cell products could be produced. Since pluripotent stem cells can be differentiated into any cell type in the three germ layers, the potential application of stem cell therapy is wide. Differentiation can be carried out ex vivo or in vivo through the transplantation of progenitor cells that continue to differentiate and mature in the organ environment of the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the procedure and ensure that the appropriate population of cells is generated before transplantation.
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3/105 [005] In most cases, however, undifferentiated pluripotent stem cells are avoided in clinical transplant therapies due to the propensity to form teratomas. On the contrary, such therapies tend to use differentiated cells (for example, cardiomyocytes derived from stem cells transplanted into the myocardium of patients suffering from heart failure). Clinical applications of such pluripotent cells or tissues would benefit from a safety feature that controls the growth and survival of cells after transplantation.
[006] The technique seeks stem cells with the ability to produce cells that are used to regenerate sick or deficient cells. Pluripotent stem cells (PSCs) can be used due to the fact that they propagate and differentiate into several possible cell types. The family of PSCs includes several members generated through different techniques and having different immunogenic characteristics. The compatibility of the patient with manipulated cells or tissues derived from PSCs determines the risk of immune rejection and the need for immunosuppression.
[007] Embryonic stem cells (CTEs) isolated from the internal cell mass of blastocysts exhibit histocompatibility antigens that are incompatible with receptors. This immune barrier cannot be resolved by CES banks of the human leukocyte antigen (HLA) type due to the fact that even the paired HLA PSC grafts undergo rejection due to mismatches in non-HLA molecules that function as minor antigens. To date, the preclinical success of PSC-based approaches has been achieved only in immunosuppressed or
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4/105 immunodeficient, or when cells are encapsulated and protected from the host's immune system. The systemic immunosuppression used in allogeneic organ transplantation, however, is not justified for regenerative approaches. Immunosuppressive drugs have serious side effects and significantly increase the risk of infections and malignancies.
[008] To circumvent the rejection problem, different techniques for the generation of patient-specific pluripotent stem cells have been developed. These include the transfer of a somatic cell nucleus to an enucleated oocyte (somatic cell nucleus transfer stem cells (SCNT)), the fusion of a somatic cell with an ESC (hybrid cell), and the reprogramming of somatic cells using certain transcription factors (induced PSCs or iPSCs). SCNT and iPSCs stem cells, however, may have immune incompatibilities with the cell nucleus or donor, respectively, despite the chromosomal identity. SCNT stem cells carry mitochondrial DNA (mtDNA) passed from the oocyte. Proteins encoded by mtDNA can act as minor relevant antigens and trigger rejection. DNA and mtDNA mutations and genetic instability associated with reprogramming and expanding the culture of iPSCs can also create minor antigens relevant to immune rejection. This previously unknown immune barrier decreases the likelihood of successful and large-scale manipulation of specific tissues from compatible patients using SCNT stem cells or iPSCs.
IV. SUMMARY OF THE INVENTION [009] Pluripotent cells (HIP) were generated
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5/105 hypoimmunes that prevent rejection by the host's immune system. Placenta syncytiotrophoblastic cells were used to form the interface between maternal blood and fetal tissue. The expression of MHC I or HLA-I and MHC II or HLAII was reduced. CD47 has been increased. This pattern of impaired antigen presentation capacity and protection of innate immune clearance prevented host immune rejection. This has been shown for HIP cells and ectoderm, mesoderm and endoderm derived cells in which the HIP cells have been differentiated.
[010] Thus, the invention provides a method for generating a hypoimmunogenic pluripotent stem cell comprising: eliminating the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); eliminate the activity of both alleles of a CIITA gene in iPSC; and increase the expression of CD47 in iPSC.
[011] In a preferred embodiment of the method, iPSC is human, the B2M gene is human, the CIITA gene is human and the increased expression of CD47 results from the introduction of at least one copy of a human CD47 gene under the control of a promoter in the iPSC cell. In another preferred embodiment of the method, iPSC is murine, the B2m gene is murine, the Cita gene is murine and the increased expression of Cd47 results from the introduction of at least one copy of a murine Cd47 gene under the control of a promoter in the iPSC cell. In a more preferred embodiment, the promoter is a constitutive promoter.
[012] In some modalities of the methods disclosed here, the disruption in both alleles of the B2M gene results from a reaction of Regularly Interspaced Grouped Short Palindromic Repeats / Cas9 (CRISPR) that disturbs both
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6/105 the alleles of the B2M gene. In other modalities of the method, the disruption in both alleles of the CIITA gene results from a CRISPR reaction that disturbs both alleles of the CIITA gene.
[013] The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more changes that inactivate both alleles of an endogenous B2M gene; one or more changes that inactivate both alleles of an endogenous CIITA gene; and one or more changes causing an increased expression of a CD47 gene in the hHIP stem cell; where the hHIP stem cell induces a lower Natural Exterminating Cell (NK) response less than a second NK cell response induced by an induced pluripotent stem cell (1PSC) comprising said B2M and CIITA changes, but not it comprises increased expression of the CD47 gene and in which the first and second responses of NK cells are measured by the ΙΕΝ-γ levels of NK cells incubated with hHIP or 1PSC in vitro.
[014] The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more changes that inactivate both alleles of an endogenous B2M gene; one or more changes that inactivate both alleles of an endogenous CIITA gene; and a change causing increased expression of a CD47 gene in the hHIP stem cell; where the hHIP stem cell induces a first T cell response in a humanized mouse strain less than a second T cell response in the humanized mouse strain induced by a 1PSC, and where the first and second T cell response are measured by determining the ΙΕΝγ levels of humanized mice in an Elispot assay.
[015] The invention provides a method, including the
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7/105 HPV stem cell transplantation, which is revealed here for a human individual. The invention further provides the use of HHH stem cells for the preparation of a medicament for treating conditions that require cell transplantations.
[016] The invention provides a hypoimmunogenic pluripotent cell that comprises an endogenous Class I Histocompatibility Antigen (HLA-I) function that is reduced when compared to a parent pluripotent cell; an endogenous function of the Class II Histocompatibility Antigen (HLA-II) that is reduced when compared to the parental pluripotent cell; and a reduced susceptibility to the death of NK cells when compared to the progenitor pluripotent cell; where the hypoimmunogenic pluripotent cell is less susceptible to rejection when transplanted into an individual as a result of reduced HLA-I function, reduced HLA-II function and reduced susceptibility to NK cell death.
[017] In some embodiments, the hypoimmunogenic pluripotent cell is reduced by a reduction in the expression of the β-2 microglobulin protein. In a preferred embodiment, a gene encoding the β-2 microglobulin protein is knocked out. In a more preferred embodiment, the β-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1. In a more preferred embodiment, the β-2 microglobulin protein has the sequence of SEQ ID NO: 1 .
[018] In some embodiments, HLA-I function is reduced by reducing the expression of the HLA-A protein. In a preferred embodiment, a gene that encodes the HLA protein
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A is subjected to knockout. In some embodiments, HLA-I function is reduced by reducing the expression of the HLA-B protein. In a preferred embodiment, a gene encoding the HLA-B protein is knocked out. In some embodiments, HLA-I function is reduced by reducing the expression of the HLA-C protein. In a preferred embodiment, a gene encoding the HLA-C protein is knocked out.
[019] In another embodiment, hypoimmunogenic pluripotent cells do not comprise an HLAI function.
[020] The invention provides a hypoimmunogenic pluripotent cell in which HLA-II function is reduced by a reduction in CIITA protein expression. In a preferred embodiment, a gene that encodes the CIITA protein is knocked out. In a more preferred embodiment, the CIITA protein has at least 90% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CIITA protein has the sequence of SEQ ID NO: 2.
[021] In some embodiments, HLA-II function is reduced by reducing the expression of the HLA-DP protein. In a preferred embodiment, a gene encoding the HLADP protein is knocked out. In some embodiments, HLA-II function is reduced by reducing the expression of the HLADR protein. In a preferred embodiment, a gene encoding the HLA-DR protein is knocked out. In some embodiments, HLA-II function is reduced by reducing the expression of the HLA-DQ protein. In a preferred embodiment, a gene encoding the HLA-DQ protein is knocked out.
[022] The invention provides hypoimmunogenic pluripotent cells that do not comprise an HLA-II function.
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9/105 [023] The invention provides hypoimmunogenic pluripotent cells with a reduced susceptibility to macrophage phagocytosis or NK cell death. The reduced susceptibility is caused by increased expression of a CD47 protein. In some embodiments, increased expression of CD47 results from a change in an endogenous CD47 gene locus. In other embodiments, the increased expression of CD47 results from a CD47 transgene. In a preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3.
[024] The invention provides hypoimmunogenic pluripotent cells that comprise a suicidal gene that is activated by a trigger that causes the hypoimmunogenic pluripotent or differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 4. In a more preferred embodiment, the HSV-tk gene encodes a protein with the sequence with SEQ ID NO: 4.
[025] In another preferred embodiment, the suicide gene is an Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC). In a more preferred embodiment, the EC-CD gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein with the sequence with SEQ ID NO: 5.
[026] In another preferred embodiment, the suicide gene encodes an inducible Caspase protein and the
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10/105 trigger is a chemical dimerization inducer (CID). In a more preferred embodiment, the inducible gene encodes the Caspase protein that comprises at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes the Caspase protein that comprises the SEQ ID sequence NO: 6. In a more preferred mode, the CID is AP1903.
[027]
The invention provides a method for producing a hypoimmunogenic pluripotent cell that comprises reducing an endogenous Class I Histocompatibility Antigen (HLA-I) function in a pluripotent cell; reduce an endogenous function of the Class II Histocompatibility Antigen (HLAII) in a pluripotent cell; and increasing the expression of a protein that reduces the pluripotent cell's susceptibility to macrophage phagocytosis or the death of NK cells.
[028]
In one embodiment of the method, the HLAI function is reduced by reducing the expression of a β-2 microglobulin protein. In a preferred embodiment, the expression of the β-2 microglobulin protein is reduced by the elimination of a gene that encodes the β-2 microglobulin protein. In a more preferred embodiment, the β-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1. In a more preferred embodiment, the β-2 microglobulin protein has the sequence of SEQ ID NO: 1 .
[029]
In another method, the function
HLA-I is reduced by reducing the expression of the HLA-A protein. In a preferred embodiment, the expression of the HLA-A protein is reduced by the knockout of a gene that encodes the HLA-A protein. In another method, the HLA-I function is reduced by reducing the expression of the HLA-B protein. In a
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11/105 preferred mode, the expression of the HLA-B protein is reduced by the knockout of a gene that encodes the HLAB protein. In another method, the HLA-I function is reduced by reducing the expression of the HLA-C protein. In a preferred embodiment, the expression of the HLA-C protein is reduced by the knockout of a gene that encodes the HLA-C protein.
[030] In another method, the hypoimmunogenic pluripotent cell does not comprise an HLA-I function.
[031] In another method, the HLA-II function is reduced by reducing the expression of a CIITA protein. In a preferred embodiment, the expression of the CIITA protein is reduced by the knockout of a gene that encodes the CIITA protein. In a more preferred embodiment, the CIITA protein has at least 90% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CIITA protein has the sequence of SEQ ID NO: 2.
[032] In another embodiment of the method, HLA-II function is reduced by reducing the expression of an HLA-DP protein. In a preferred embodiment, the expression of the HLA-DP protein is reduced by the knockout of a gene that encodes the HLA-DP protein. In another modality of the method, HLA-II function is reduced by reducing the expression of an HLA-DR protein. In a preferred embodiment, the expression of the HLA-DR protein is reduced by the knockout of a gene that encodes the HLA-DR protein. In some modalities of the method, HLA-II function is reduced by reducing the expression of an HLA-DQ protein. In a preferred embodiment, the expression of the HLA-DQ protein is reduced by the knockout of a gene that encodes the HLA-DQ protein.
[033] In another method, the cell
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12/105 hypoimmunogenic pluripotent does not comprise an HLA-II function.
[034] In another method, the increased expression of a protein that reduces the pluripotent cell's susceptibility to macrophage phagocytosis results from a change in an endogenous gene locus. In a preferred embodiment, the endogenous gene locus codes for the CD47 protein. In another embodiment, the increase in protein expression results from the expression of a transgene. In a preferred embodiment, the transgene encodes a CD47 protein. In a more preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3.
[035] Another modality of the method also comprises expressing a suicide gene that is activated by a trigger that causes the pluripotent or hypoimmunogenic differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 4. In a more preferred embodiment, the HSV-tk gene encodes a protein with the sequence with SEQ ID NO: 4.
[036] In another modality of the method, the suicide gene is an Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC). In a preferred embodiment, the EC-CD gene encodes a protein with at least 90% sequence identity with SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein with the sequence with SEQ ID NO: : 5.
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13/105 [037] In another method, the suicide gene encodes an inducible Caspase protein and the trigger is a specific chemical dimerization inducer (ICD). In a preferred embodiment of the method, the gene encodes an inducible caspase protein that comprises at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes the inducible Caspase protein that comprises the sequence of SEQ ID NO: 6. In a more preferred embodiment, the CID is AP1903.
V. BRIEF DESCRIPTION OF THE DRAWINGS [038] Figure IA shows the rational analysis for the new hypoimmune pluripotent cells described here. Fetuses are protected from rejection during pregnancy by fetal-maternal tolerance. The cells have negative MHC class I expression. They also have a negative expression of MHC class I. They also have CD47 positively regulated. Figure 1B shows that fetal-maternal tolerance is mediated by syncytiotrophoblastic cells. Figure 1C shows that syncytiotrophoblastic cells do not have MHC I and II and elevated levels of CD47.
[039] Figure 2 shows induced murine pluripotent stem cells (miPSC) generated from C57BL / 6 fibroblasts. Pluripotency was demonstrated by the polymerase chain reaction via reverse transcriptase (rtPCR). Multiple MRNAs associated with pluripotency have been detected in extracts from miPSC cells, but not in non-induced cells (murine parental fibroblasts).
[040] Figure 3 confirms the pluripotency of miPSC cells. MiPSC C57BL / 6 cells formed teratomas in singenic mice as well as nude and beige BALB / c mice
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14/105 clear. No teratomas were formed in immunocompetent allogeneic BALB / c mice.
[041]
Figure 4 shows that when β-2-microglobulin expression is eliminated in miPSC cells, MHC-I expression cannot be induced by IFN-γ stimulation (right panel). As a control, the parental miPSC cells were stimulated with IFN-γ (left panel) and increased their MHC-I expression.
[042]
Figure 5 shows that the miPSC / B-2-microglobulin knockout which further comprises a Ciita expression knockout (double knockout) did not show any expression of base MHC-II and cannot be induced by TNF-a to express MHC -II.
[043]
Figure 6A shows the increased expression of Cd47 from a transgene added to the β-2-microglobulin / Ciita double knockout ( ^hypo iPSC cells). Figure 6B shows that C57BL / 6 iPSC ^hi P ° cells survive in the BALB / c allogeneic environment, but parental iPSC cells do not.
[044]
Figure 7 shows an embodiment of the invention. It shows a schematic diagram of the manipulation of iPSC that resulted in the hypoimmune pluripotent cells of the invention. To generate hypoimmune stem cells, first CRISPR-Cas 9 was used to eliminate both B2m alleles. Second, CRISPR-Cas 9 manipulation was used to eliminate both alleles of the Ciita gene. Third, a lentivirus was used to knockin a Cd47 gene.
[045]
Figure 8A schematically shows the role of B2m in the MHC I complex. A B2m knockout depletes MHC I in mice or HLA-I in humans. Figure 8B shows schematically that Ciita is a transcription factor
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15/105 that causes MHC II expression in mice or HLA-II expression in humans. A Ciita knockout depletes expression of MHC II or HLA-II.
[046] Figures 9A, 9B and 9C show that B2m- / iPSCs lack expression of MHC-I, B2m - / - Ciita - / - iPSCs lack MHC-I and MHC-II and B2m - / - Ciita- / - Cd47 tg iPSCs lack MHC-I and MHC-II and overexpress Cd47.
[047] Figures 10A, 10B, 10C, 10D and 10E show mouse models of transplanted vs. wild-type iPSCs. Hypoimmune PSCs in allogeneic or syngenic host mice. Here, iPSCs were formed from C57BL / 6 mice, and allogeneic mice are BALB / c. Figure 10A, wild-type iPSCs formed only teratomas in the thighs of isogenic C57BL / 6 mice. In contrast, an immune response was mounted in the allogeneic host mice (BALB / c) and no teratoma grew. Figure 10B, wild-type iPSCs formed only teratomas in isogenic C57BL / 6 mice. In Figure 10C, the immune response prevented the formation of teratoma in allogeneic BALB / c. Figure 10D compares the response of T cells (IFN-γ and IL-4) to iPSC in syngeneic and allogeneic hosts using a point frequency assay (frequency of cells that release IFN-γ and IL-4). The release of IFN-γ and IL-4 was very low in C57BL / 6 hosts, but increased dramatically in BALB / c hosts. Figure 10E represents the responses of B cells in syngeneic and allogeneic hosts. The iPSCs were incubated with the serum of the host animals that previously received iPSCs. Bound immunoglobulins were measured using flow cytometry. The mean fluorescence intensity (MFI) was significantly higher for serum collected in
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16/105 allogeneic BALB / c receptor hosts.
[048] Figures 11A, 11B, 11C, 11D and 11E show the partial effect of the knockout of the B2m gene on the iPSCs described above. Figure 11A, B2m - / - iPSCs grew on isogenic C57BL / 6 mouse thighs, forming teratomas due to lack of immune response, while a partial immune response was mounted in the host's allogeneic mice (BALB / c); for example, some of the transplanted cells survive. The IPSCs in Figure 11B, B2m - / - formed teratomas in the syngeneic mice. Figure 11C, partial survival (60%) was achieved in allogeneic hosts. In Figure 11D, the differences in the T cell response (ΙΕΝ-γ and IL-4) between the two hosts showed that a smooth but detectable T cell response against B2m - / - IPSCs. Figure 11E shows the responses of B cells in the different host mice, showing the weaker immune response when compared to wild-type IPSCs. There was a significantly stronger immunoglobulin response after the allogeneic transplantation of B2m - / - IPSCs in BALB / c when compared to the syngeneic transplantation in C57BL / 6. Thus, there was a limited survival of IPSCs B2m - / - in allogeneic recipients.
[049] Figures 12A, 12B, 12C, 12D and 12E show the increase in the partial effect of the knockout of the B2m gene and the Ciita gene on iPSCs on cell survival in single and allogeneic host mice. The IPSCs of Figure 12A, B2m - / - Ciita - / - formed teratomas in the thighs of C57BL / 6 mice due to the lack of immune response, while a partial immune response (but reduced when compared to the B2m - / - immune response) was mounted on the mice
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17/105 allogeneic host (BALB / c). Figure 12B, B2m - / - Ciita / - iPSCs formed teratomas in the syngeneic mouse. Figure 12C shows that some cell grafts (91.7%) survive in allogeneic hosts. Figure 12D, the differences in T cell responses (IFN-γ and IL-4) between the two hosts showed a slightly higher IFN-γ response in allogeneic versus syngenic receptors. Figure 12E represents the responses of B cells in the different host mice. The weaker immune response was compared with iPSCs by weight and B2m - / - iPSCs. A significant difference between allogeneic and syngeneic receptors was not observed. In general, there was limited survival of B2m- / Ciita - / - iPSCs in allogeneic receptors that can be attributed to a measurable immune response.
[050] Figures 13A, 13B, 13C, 13D and 13E show the effect of the knockout of the B2m gene and the Ciita gene and of the knockout in the Cd47 transgene in iPSCs on cell survival in singenic and allogeneic host mice. Figure 13A, B2m - / - Ciita - / - Cd47tg iPSCs teratomas grew on thighs of a single and allogeneic C57BL / 6 host. 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 response of T cells (IFN-γ and IL-4) in allogeneic receptors. No difference between the two hosts was observed. Figure 13E represents the lack of B cell responses in allogeneic receptors. No difference between the two hosts was observed. Thus, there was complete survival of B2m- / Ciita - / - Cd47tg iPSCs in allogeneic recipients. They do not
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18/105 were immunogenic, as they did not elicit T cell or B cell response.
[051] Figures 14A, 14B and 14C show that B2m - / - Ciita - / - Cd47tg iPSCs (referred to as non-immunogenic pluripotent cells (HIP)) evaded the host's immune system. Figure 14A, the expression of NK stimulator cells did not increase in HIP cells. A fusion protein that recognizes various NKG2D cell transmembrane protein ligands has been used to assess the level of activating ligands, which can activate the activity of cytolytic NK cells. The binding of fusion protein to IPSCs is, therefore, a general parameter for its expression of NKG2D ligand activation. In Figure 14B, HIP cells did not increase the expression of CD107a from NK cells, a marker of the functional activity of NK cells. In contrast, B2m - / - Ciita- / iPSCs induced the expression of CD107a in cells and, thus, triggered their cytolytic function. Figure 14C, ΙΕΝ-γ Elispot assays with purified NK cells purified from C57BL / 6 mouse spleen did not show NK cell response induced by HIP cells. Thus, NK cells were not activated to release ΙΕΝ-γ. The spot frequency for HIP cells was not different from that of unstimulated NK cells (neg control). Only B2m - / - Ciita - / - IPSCs resulted in significantly increased spot customers of ΙΕΝ-γ.
[052] Figures 15A and 15B show additional data showing that HIP cells prevent rejection or death by the innate immune system due to the Cd47 transgene. An in vivo NK cell assay had a mixture of 50% iPSCs and 50% HIPs that were injected into the NK-rich peritoneum of isogenic C57BL / 6 (singenic) mice. Agui, a
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19/105 cytotoxicity is caused by NK cells. After 24 and 48 hours, the peritoneal cells were recovered and separated. Figure 15A compares the iPSCs with B2m - / - Ciita - / - iPSCs (without Cd47 transgene). B2m - / - Ciita - / - iPSCs were selectively killed by NK cells. Figure 15B compares iPSCs with B2m - / - Ciita / - Cd47 tg iPSCs (HIP cells). HIP cells were not selectively killed by NK cells. The 50% ratio of HIP cells between the peritoneal iPSCs was maintained, indicating no stimulation of NK cells. Thus, while the MHC-I and MHC-II knockouts made the cells highly susceptible to the death of NK cells, the overexpression of Cd47 removed the stimulatory NK cell interaction.
[053] Figure 16 shows that the murine HIP cells of the invention exhibited a normal murine karyotype.
[054] Figures 17A, 17B and 17C show that the murine HIP cells of the invention retained pluripotency during the manipulation process. Rt-PCR analysis of generally accepted markers to indicate pluripotency is shown (Nanog, Oct 4, Sox2, Esrrb, Tbx3, Tcll and actin as load control). Pluripotent markers were expressed throughout the three-stage manipulation process. Figure 17A compares iPSCs, B2m - / - iPSCs and murine fibroblasts (negative control). B2m - / - iPSC cells retained pluripotency genes. Figure 17B shows the same analysis, but the B2m - / - Ciita - / - iPSCs. They retained the same pluripotency genes. Figure 17C shows the same analysis, but with B2m - / - Ciita - / - Cd47 tg iPSCs (HIP cells). These cells retained the same pluripotency genes. In addition, histological images of teratomas that developed after HIP cell transplantation in beige SCID mice show
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20/105 that the cell types associated with ectoderm, mesoderm and endoderm were identified. Immunofluorescence markers for all three germ layers were detected (data not shown). Cell morphology was correct for neuroectoderm, mesoderm and endoderm. Immunofluorescence staining for DAPI, GFAP, cytokeratin 8 and brachyuria confirmed the pluripotence of HIP cells.
[055] Figures 18A, 18B show HIP cells differentiated into cells of the mesodermal lineage and have lost their pluripotency markers. Figures 18A shows that pluripotent markers in HIP cells (marked with mHIP) were lost in differentiated murine endothelial cells (marked with miEC). Figures 18B shows that pluripotent markers were retained in HIP cells, but not in differentiated murine smooth muscle cells (marked with miSMC). Figures 18C shows that pluripotent markers were retained in HIP cells, but not in cells of differentiated murine cardiomyocytes (marked with miCM). These results were confirmed by immunohistochemistry (data not shown). Endothelial cells were detected using anti-CD31 and anti-VE-cadherin, smooth muscle cells were detected using anti-SMA and anti-SM22 antibodies, and cardiomyocytes were detected with anti-Troponin I and anti-sarcomeric alpha actinin antibodies.
[056] Figures 19A and 19B show that HIP cells were differentiated into islet cells of endoderm lineage (ilCs) that produced C-peptide and insulin. Figure 19A, differentiation markers were not detected in HIP cells, but were in the induced islet cells.
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Figure 19B, the induced islet cells produced insulin. Immunohistochemistry staining for peptide C confirmed these results (data not shown).
[057] Figures 20A and 20B show the differentiated HIP cells in the ectoderm lineage. Figure 20A shows the HIP cells in vitro and Figure 20B shows the differentiated neuronal cells. Immunohistochemical staining with the neuroectodermal stem cell marker Nestin and Tuj-1 confirmed these results (data not shown).
[058] Figures 21A, 21B and 21C show that cells differentiated from HIP cells retained the MHC I and II phenotype and Cd47 overexpression. Figure 21A compares the expression of MHC-I, MHC-II and Cd47 between mouse induced endothelial cells (miEC) and B2m - / - Ciita- / Cd47 tg miEC cells. Figure 21B compares the expression of MHC-I, MHCII and Cd47 between mouse-induced smooth muscle cells (miSMC) and B2m - / - Ciita - / - Cd47 tg mSMC cells. Figure 21C compares the expression of MHC-I, MHC-II and Cd47 between mouse-induced myocardocytes (miCM) and B2m - / - Ciita - / - Cd47 tg miMC cells.
[059] Figures 22A, 22B and 22C show that endothelial cells differentiated from HIP cells are nonimmunogenic. Figure 22A, transplantation of singenic and allogeneic miCs mice C56BL / 6. miECs in allogeneic BALB / c receptor mice generated a pronounced immune response, but not in syngeneic mice. This was evidenced by strong responses of ΙΕΝ-γ Elispot and immunoglobulin (FACS analysis) at BALB / c receptors (Figure 22B). Figure 22C, neither HIP nor miEC cells generated an immune response at syngeneic or allogeneic receptors.
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22/105 [060] Figures 23A, 23B and 23C show that mouse-induced smooth muscle cells differentiated from HIP cells are non-immunogenic. Figure 23A, transplantation of singenic and allogeneic mice of C56BL / 6 miSMCs. miSMCs in allogeneic BALB / c receptor mice generated a pronounced immune response, but not in syngeneic mice. This was evidenced by strong responses of IFN-γ Elispot and immunoglobulin (FACS analysis) at BALB / c receptors. Figure 23C, neither HIP nor miSMC cells generated an immune response in syngeneic or allogeneic receptors.
[061] Figures 24A, B and C show that mouse-induced cardiomyocyte cells differentiated from HIP cells are non-immunogenic. Figure 25A, transplantation of single and allogeneic C56BL / 6 miCMCs. Figure miCMCs in allogeneic BALB / c receptor mice generated a pronounced immune response, but not in singenic mice. This was evidenced by strong responses of ΙΕΝ-γ Elispot and immunoglobulin (FACS analysis) in BALB / c receptors (Figure 24B). Figure 24C, neither HIP nor miCMC cells generated an immune response at syngeneic or allogeneic receptors.
[062] Figure 25 shows that differentiated cells (miECS, miSMCs, miCMs) derived from HIP cells prevent rejection via the innate immune system. An NK fusion protein assay showed that none of the three differentiated cells showed increased expression of NK stimulating cell ligands when compared to differentiated cells derived from miPSCs.
[063] Figures 26A and 26B show that miECs derived from HIP cells of the invention prevented the immune reaction and achieved long-term survival in a host
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23/105 allogeneic. Figure 26A, miEC grafts derived from miPSCs showed long-term survival in syngeneic receptors (C57BL / 6), but were rejected in allogeneic receptors (BALB / c). Figure 26B, HIP-derived miECs achieve long-term survival after transplantation into syngeneic and allogeneic recipients.
[064] Figure 27: miECS derived from HIP cells organized to form vascular structures in allogeneic hosts. After transplantation into a Matrigel matrix, over six weeks, the miECs were organized in a three-dimensional manner to form vascular structures. These results were confirmed by immunofluorescence for luciferase and VE-cadherin; miECs were transduced to express luciferase prior to transplantation. Survival was monitored through bioluminescence imaging and transplanted cells were identified with immunofluorescence staining against luciferase (data not shown).
[065] Figure 28 shows that human HIP cells exhibited a normal human karyotype.
[066] Figures 29 show that human HIP cells maintained their pluripotency during the manipulation process. HiPSCs (for example, the starting cells, before the changes of the invention) and the HIP cells of the invention have expression of the pluripotency genes (NANOG, OCT4, SOX2, DPPA4, hTERT, ZFP42 and DEMT3B; G3PDH served as load control ) using PCR assays. Immunofluorescent staining confirmed this result, since cells express TRA-1-60, TRA-1-81, Sox2, Oct4, SSEA-4 and alkaline phosphatase markers (data not shown).
[067] Figures 30A and 30B show that the cells
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Human HIPs transplanted into humanized allogeneic mice did not cause an immune response. Figure 30A shows that T cells did not respond to transplanted HIP cells, as measured by the production of IFN-γ or IL5 in the Elispot assays. In return, the transplanted CEPi did so. Figure 30B shows that only iPSCs caused a strong antibody response in flow cytometry. HIP cells did not.
[068] Figures 31A, 31B, 31C and 31D show that human HIP cells were differentiated into the mesodermal lineage. Figure 31A shows the morphology of a human HIP cell. Figure 31B shows HIP-derived endothelial cells stained with CD31, VE-cadherin and DAPI as a control. Figure 31C shows the HIP-derived cardiomyocytes stained with α-sarcomeric actinin, Troponin I and DAPI as a control. Figure 31D shows the premature vessel formation by HIP-derived endothelial cells. HIP-derived cardiomyocytes were observed by tapping (data not shown).
[069] Figures 32A and 32B show that transplanted human endothelial cells derived from human HIP cells did not cause an immune response in allogeneic humanized mice. Figure 32A, hiECs mounted a significant T cell response in the Elispot IFN-γ and IL5 assays, while hiECs derived from human HIP cells did not. Figure 32B shows the response of B cells in flow cytometry. Only hiECs generated a significant immunoglobulin binding, as measured by mean fluorescence intensity (MFI).
[070] Figures 33A and 33B show that the
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25/105 transplantation of human cardomyocytes derived from human HIP cells did not result in an immune response in humanized allogeneic mice. Figure 33A shows the differences in T cell responses to wild-type hiCMs versus B2M - / - CIITA - / - CD47tg HIP cells in IFN-γ and IL5 ELispots. Figure 33B shows the response of B cells in flow cytometry. Only wild-type hiCMs generated a significant load of hiEC immunoglobulins, as measured by mean fluorescence intensity (MFI).
[071] Figures 34A, 34B, 34C and 34D show that the human HIP cells of the invention prevented the rejection of the innate immune system. NK cells were isolated from BALB / c mice using Magnetically Activated Cell Classification (MACS). 5X106 stimulator cells (derived from C57BL / 6 iPSC, iEC, iSMC or iCM and B2M - / - CIITA - / - or B2M - / - CIITA / - CD47 tg), were incubated with 5X106 NK cells classified by MACS on a plate of IFN-γ Elispot. After 24 hours, the spot frequency was determined with an Elispot reader. All three B2M derivatives - / - CIITA - / - induced a strong NK response. All three B2M - / - CIITA- / CD47 derivatives, however, did not elicit any response from NK cells and their spot frequency was not statistically different from negative controls (isolated NK cells not incubated with a stimulator cell). Figure 34A shows endothelial cells. Figure 34B shows smooth muscle cells. Figure 34C shows cardiomyocytes. Figure 34D shows the positive control of YAC-1 mouse lymphoma.
[072] Figures 35A, 35B and 35C show the innate immune response (or lack thereof). A mixture of 50% by weight of derivative (5X10 ⁶ cells) and 50% or more was prepared
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C57BL / 6 B2m - / - Ciita - / - or derived from B2m - / - Ciita - / - Cd47 tg (5X10 ⁶ cells). The cells were stained with 10 μΜ CFSE staining for 10 min and resuspended in 500 μΐ of saline. The cell mixture was then injected into the NK-rich peritoneum of C57BL / 6 mice (syngeneic). In this syngeneic model, all cytotoxicity is caused by NK cells. After 48 h, the peritoneal cells were recovered and their ratio was calculated. The wt and manipulated cells were identified by MHCI staining in FACS. Figure 35A shows endothelial cells. Figure 35B shows smooth muscle cells. Figure 35C shows cardiomyocytes.
[073] Figures 36A, 36B and 36C show the genetic manipulation of human iPSCs verified by FACS. The lack of HLA I and HLA II was confirmed in B2M - / - CIITA- / hiSCs. Additionally, B2M - / - CIITA - / - CD47 tg showed high expression of CD47. Figure 36A shows the results of HLA I. Figure 36B shows the results of HLA II. Figure 36C shows the CD47 results.
[074] Figures 37A and B show that the immune phenotype was maintained after differentiation from B2M - / - CIITA - / - CD47 tg iPSCs. When compared with unmodified wt derivatives, FACS analysis showed that B2M - / - CIITA- / CD47 tg derivatives lacked HLA I and HLA II and CD47 overexpression. Figure 37A shows endothelial cells and Figure 37B shows cardiomyocytes.
SAW. DETAILED DESCRIPTION OF THE INVENTION
A. INTRODUCTION [075] The invention provides hypoimmunogenic pluripotent cells (HIP) that prevent host immune responses due to various genetic manipulations, such as
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27/105 described here. The cells lack important immune antigens that elicit immune responses and are designed to prevent phagocytosis. This allows for the derivation of ready-to-use cellular products to generate specific tissues and organs. The benefit of having the ability to use derivatives of human allogeneic HIP cells in human patients results in significant benefits, including the ability to avoid long-term adjunct immunosuppressive therapy and the use of drugs commonly seen in allogeneic transplants. It also provides significant cost savings, as cell therapies can be used without the need for individual treatments for each patient. Recently, it has been shown that cell products generated from autologous cell sources can become subject to immune rejection with few or even a single antigenic mutation. Thus, autologous cellular products are not inherently non-immunogenic. In addition, cell engineering and quality control are very laborious and expensive and autologous cells are not available for acute treatment options. Only allogeneic cell products can be used for a larger patient population if the immune obstacle can be overcome. HIP cells will serve as a universal cell source for the generation of universally acceptable derivatives.
[076] The present invention concerns the exploration of the fetal-maternal tolerance that exists in pregnant women. Although half of the human leukocyte antigens (HLA) of the fetus are inherited paternally and the fetus expresses HAP antigens with greater disparities, the maternal immune system does not recognize the fetus as an allogeneic entity and does not initiate an immune response, for example, as seen in a kind of reaction
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28/105 host versus graft immune. Fetal-maternal tolerance is primarily mediated by syncytiotrophoblastic cells at the maternal-fetal interface. As shown in Figure 7, syncytiotrophoblast cells show little or no protein from major histocompatibility complexes I and II (MHC-I and MHC-II), as well as increased expression of CD47, known as the protein don't eat me that suppresses innate phagocytic immune surveillance and elimination of cells without HLA. Surprisingly, the same tolerogenic mechanisms that prevent fetal rejection during pregnancy also allow the invention's HIP cells to escape rejection and facilitate long-term survival and grafting of these cells after allogeneic transplantation.
[077] These results are additionally surprising in that this fetal-maternal tolerance can be introduced with just three genetic modifications (compared to the starting iPSCs, for example, hiPSCs), two reductions in activity (knockouts as described here) and an increase in activity (a knockin '' as described here). Generally, others skilled in the art tried to suppress the
immunogenicity of iPSCs, but were only partially successful successful; see Rong et al., Cell Stem Cell 14: 121-130 (2014) and Gornalusseet al., Nature Biotech doi: 10.1038 / nbt.3860).[078] So, The invention provides the generation of
HIP cells from pluripotent stem cells and then their maintenance, differentiation and finally transplantation of their derivatives in patients in need of them.
B. DEFINITIONS [079] The term pluripotent cells refers to
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29/105 to cells that can self-renew and proliferate while remaining in an undifferentiated state and that, under the right conditions, can be induced to differentiate into specialized cell types. The term pluripotent cells, as used herein encompasses embryonic stem cells and other types of stem cells, including fetal, amnionic or somatic stem cells. Exemplary human stem cell lines include the human embryonic stem cell line H9. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human embryonic Stem Cell Stem and the Howard Hughes Medical Institute HUES collection (as described in Cowan, CA et. Al, New England J. Med. 350: 13 (2004), here incorporated by reference in its entirety.) [080] Pluripotent stem cells, as used here, have the potential to differentiate into any of the three germ layers: endoderm (for example, stomach connection , gastrointestinal tract, lungs, etc.), mesoderm (for example, muscle, bone, blood, urogenital tissue, etc.) or ectoderm (for example, epidermal and nervous system tissues). The term pluripotent stem cells, as used herein, also encompasses induced pluripotent stem cells, or iPSCs, a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of progenitor cells include somatic cells that have been reprogrammed to induce a pluripotent undifferentiated phenotype, by various means. Such iPS or iPSC cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for
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30/105 IPS cell induction are known in the art and are further described below. (See, for example, Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Blotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8: 381-384 (2009); each is incorporated by reference in its entirety.) A generation of pluripotent stem cells induced (IPSCs) is described below. As used herein, hiPSCs are human induced pluripotent stem cells and miPSCs are murine induced pluripotent stem cells.
[081] Characteristics of pluripotent stem cells refer to characteristics of a cell that distinguishes pluripotent stem cells from other cells. The ability to originate progenies that can undergo differentiation, under the appropriate conditions, in cell types that collectively demonstrate characteristics associated with cell lines of all three germ layers (endoderm, mesoderm and ectoderm) is a characteristic of pluripotent stem cells. Expression or non-expression of certain combinations of molecular markers are also characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least several and, in some embodiments, all the markers in the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA- 2-49 / 6E, ALP, Sox2, E-cadherin, UTF1, Oct4, Rexl and Nanog. Cellular morphologies associated with pluripotent stem cells are also characteristic of pluripotent stem cells. As described here, cells do not need to go through pluripotency to be
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31/105 reprogrammed into endodermal progenitor cells and / or hepatocytes.
[082] As used herein, multipotent or multipotent cell refers to a type of cell that can give rise to a limited number of other types of particular cells. For example, multipotent cells have the ability to form endodermal cells. In addition, multipotent blood stem cells can differentiate into various types of blood cells, including lymphocytes, monocytes, neutrophils, etc.
[083] As used here, the term oligopotent refers to the ability of an adult stem cell to differentiate into just a few different cell types. For example, lymphoid or myeloid stem cells have the ability to form cells of lymphoid or myeloid lineages, respectively.
[084] As used here, the term unipotent means the ability of a cell to form a single cell type. For example, sperm stem cells only have the ability to form sperm.
[085] As used here, the term totipotent means the ability of a cell to form an entire organism. For example, in mammals, only the blastomeres of the zygote and the first stage of divage are totipotent.
[086] As used here, non-pluripotent cells refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, without limitation, cells from a selected tissue of bone marrow, skin, skeletal muscle,
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32/105 adipose tissue and blood. Exemplary cell types include, without limitation, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts and T cells. The starting cells used to generate the multipotent induced cells, the endodermal progenitor cells and the hepatocytes can be non-pluripotent cells.
[087] Differentiated cells include, without limitation, multipotent cells, oligopotent cells, unipotent cells, progenitor cells and terminally differentiated cells. In particular embodiments, a less powerful cell is considered to be differentiated in reference to a more powerful cell.
[088] A somatic cell is a cell that forms the body of an organism. Somatic cells include cells that form organs, skin, blood, bones and connective tissue in an organism, but not germ cells.
[089] The cells can be, for example, human or non-human mammals. Exemplary non-human mammals include, without limitation, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle and non-human primates. In some embodiments, a cell is from an adult human or non-human mammal. In some embodiments, a cell is from a neonatal human, an adult human, or a non-human mammal.
[090] As used here, the terms individual or patient refer to animal gualguer, such as a domesticated animal, a zoo animal or a human being. The individual or patient can be a mammal such as a dog, cat, bird, cattle or a human being. Specific examples of individuals and patients include, without
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33/105 limitation, individuals (particularly humans) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow and the like.
[091] Mammalian cells can be human or non-human mammals. Exemplary non-human mammals include, without limitation, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle and non-human primates (for example, chimpanzees, monkeys and baboons).
[092] A hypoimmunogenic pluripotent cell or HIP cell here means a pluripotent cell that retains its pluripotent characteristics and gives rise to a reduced immune rejection response when transferred to an allogeneic host. In preferred embodiments, HIP cells do not generate an immune response. Thus, hypoimmunogenic refers to a significantly reduced or eliminated immune response when compared to the parental immune response cell (i.e., wt) before immunomanipulation, as presented herein. In many cases, HIP cells are immunologically silent and still retain pluripotent capabilities. The tests for the characteristics of the HIP are described below.
[093] The HLA complex or human leukocyte antigen is a gene complex that encodes the proteins of the major histocompatibility complex (MHC) in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two MHCs, class I and class II, HLA-I and HLA-II. HLA-I includes three proteins,
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HLA-A, HLA-B and HLA-C, which have peptides from inside the cell, and antigens presented by the HLA-I complex attract exterminating T cells (also known as CD8 + T cells or cytotoxic T cells). HLA-I proteins are associated with β-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4 + cells (also known as cells Auxiliary tees). It should be understood that the use of MHC or HLA is not intended to be limiting, as it depends on whether the genes are from humans (HLA) or murine (MHC). Thus, with respect to mammalian cells, these terms can be used interchangeably in this document.
[094] By gene knockout we mean a process that makes a particular gene inactive in the host cell in which it resides, resulting in no proteins of interest being produced or in an inactive form. As will be appreciated by those skilled in the art and described below, this can be accomplished in several different ways, including removing nucleic acid sequences from a gene, or interrupting the sequence with other sequences, changing the reading frame or changing the regulatory components of nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with meaningless sequences, all or part of a regulatory sequence, such as a promoter, can be removed or replaced, translation initiation sequences can be removed or replaced, etc.
[095] By gene knockin we mean a process that adds a genetic function to a cell
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35/105 hostess. This causes increased levels of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in a number of ways, including adding one or more additional copies of the gene to the host cell or changing a regulatory component of the endogenous gene, increasing the expression of the protein. This can be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.
[096] The β-microglobulin or β2Μ or B2M protein refers to the human β2Μ protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000015.10: 44711487-44718159.
[097] The CD47 protein refers to the human β2Μ protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000016.10: 10866208-10941562.
[098] The CIITa protein refers to the human CIITA protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000003.12: 108043094-108094200.
[099] By wild type in the context of a cell means a cell found in nature. However, in the context of a pluripotent stem cell, as used herein, it also means that iPSC which may contain nucleic acid changes resulting in pluripotency, but has not undergone the genetic editing processes of the invention to achieve hypoimmunogenicity.
[0100] By singular here it refers to the similarity or genetic identity of a host organism
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36/105 and cell transplantation where there is immunological compatibility; for example, no immune response is generated.
[0101] By allogeneic here it refers to the genetic dissimilarity of a host organism and cell transplant where an immune response is generated.
[0102] By B2M - / - here means that a diploid cell had the B2M gene inactivated on both chromosomes. As described here, this can be accomplished in a variety of ways.
[0103] By CIITA - / - here means that a diploid cell had the CIITA gene inactivated on both chromosomes. As described here, this can be accomplished in a variety of ways.
[0104] By CD47 tg (represented by transgene) or CD47 +) it is understood that this host cell expresses CD47, in some cases, having at least one additional copy of the CD47 gene.
[0105] An Oct polypeptide refers to any of the Octamer families of naturally occurring transcription factors, or variants thereof, that maintain similar transcription factor activity (within at least 50%, 80% or 90% of activity) compared to the nearest naturally occurring family member or polypeptides that comprise at least the DNA binding domain of the naturally occurring family member, and may further comprise a transcription activation domain. Exemplifying Oct polypeptides include Oct-1, Oct-2, Oct3 / 4, Oct-6, Oct-7, Oct-8, Oct-9 and Oct-11. Oct3 / 4 (referred to herein as Oct4) contains the POU domain, at a 150 amino acid sequence conserved between Pit-1, Oct-1, Oct-2 and uric-86.
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37/105 (See, Ryan, AK & Rosenfeld, MG, Genes Dev. 11: 12071225 (1997), incorporated by reference in its entirety.) In some embodiments, variants are at least 85%, 90% or 95% of amino acid sequence identity in its complete sequence compared to a naturally occurring Oct polypeptide family member, such as those listed above or as listed under Genbank accession number NP002692.2) or NP-038661.1 (mouse Oct4). The Oct polypeptides (for example, Oct3 / 4 or Oct 4) can be from humans, mice, rats, cattle, pigs or other animals. Generally, the same protein species will be used with the manipulated cell species. The Oct polypeptide (or polypeptides) can be a pluripotency factor that can help induce multipotency in non-pluripotent cells.
[0106] A Klf polypeptide refers to any of the naturally occurring members of the Krüppel family of factors (Klfs), zinc finger proteins that contain amino acid sequences similar to those of the Drosophila Krüppel embryo pattern regulator, or variants of naturally occurring members that maintain similar activity factor transcription (within at least 50%, 80% or 90% activity) compared to the nearest related naturally occurring family member, or polypeptides that comprise at least the domain of DNA binding of the naturally occurring family member, and may further comprise a transcriptional activation domain. (See, Dang, DT, Pevsner, J. & Yang, VW, Cell Biol. 32: 1103-1121 (2000), hereby incorporated by reference in their entirety.) Exemplary Klf family members include, Klfl, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, KlflO, Klfll, Klfl2,
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Klfl3, Klfl4, Klfl5, Klfl6 and Klfl7. It was found that Klf2 and Klf-4 were factors with the capacity to generate IPS cells and the related genes Klfl and Klf5 as well, although with reduced efficiency. (See, Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), hereby incorporated by reference in its entirety.) In some embodiments, variants have at least 85%, 90% or 95% identity of amino acid sequence in its complete sequence compared to a naturally occurring member of the Klf polypeptide family, such as those listed above or as listed under Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (for example, Klfl, Klf4 and Klf5) can be from humans, mice, rats, cattle, pigs or other animals. Generally, the same protein species will be used with the manipulated cell species. The Klf polypeptide (or polypeptides) can be a pluripotency factor. Expression of the Klf4 gene or polypeptide can help induce multipotency in an initial cell or in a population of initial cells.
[0107] A Myc polypeptide refers to any of the naturally occurring members of the Myc family. (See, for example, Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6: 635-645 (2005), hereby incorporated by reference in their entirety.) It also includes variants that maintain an activity similar to the transcription factor when compared to the nearest naturally occurring family member (that is, with an activity of at least 50%, 80% or 90%).
Additionally, it includes polypeptides that comprise at least the DNA binding domain of a naturally occurring family member, and may additionally comprise a
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39/105 transcriptional activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc and L-Myc.
In some modalities, the variants have at least 85%, 90% or 95% of amino acid sequence identity in its sequence complete compared to a family member
naturally occurring Myc polypeptide, such as those listed above or as listed under Genbank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) can be from humans, mice, rats, cattle, porcines or other animals. Generally, the same protein species will be used with the manipulated cell species. The Myc polypeptide (or polypeptides) can be a pluripotency factor.
[0108] A Sox polypeptide refers to any of the naturally occurring members of the SRM-related HMG-box (Sox) transcription factors, characterized by the presence of the high mobility group (HMG) domain, or variants thereof, that maintain similar transcription factor activity when compared to the nearest related natural family member (that is, with at least 50%, 80% or 90% activity). In addition, it includes polypeptides that comprise at least the DNA-binding domain of the naturally occurring family member, and may further comprise a transcriptional activation domain. (See, for example, Dang, DT et al., Int. J. Biochem. Cell Biol. 32: 1103-1121 (2000), hereby incorporated by reference in its entirety.) Exemplary Sox polypeptides include, for example, Soxl, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, SoxlO, Soxll, Soxl2, Soxl3, Soxl4, Soxl5, Soxl7, Soxl8, Sox-21 and Sox30. Soxl has been shown to produce IPS cells with an efficiency similar to Sox2, and
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40/105 the Sox3, Soxl5 and Soxl8 genes have also been shown to generate IPS cells, although slightly less efficiently than Sox2. (See, Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), hereby incorporated by reference in its entirety.) In some embodiments, variants have at least 85%, 90% or 95% identity of amino acid sequence in its complete sequence compared to a member of the naturally occurring Sox polypeptide family such as those listed above or listed under Genbank accession number CAA83435 (human Sox2). The Sox polypeptides (for example, Soxl, Sox2, Sox3, Soxl5 or Soxl8) can be from humans, mice, rats, cattle, pigs or other animals. Generally, the same protein species will be used with the manipulated cell species. The Sox polypeptide (or polypeptides) can be a pluripotency factor. As discussed here, SOX2 proteins find particular use in the generation of iPSCs.
[0109] By differentiated hypoimmunogenic pluripotent cells or differentiated HIP cells or dHIP cells here are iPS cells that have been engineered to have hypoimmunogenicity (for example, by eliminating B2M and CIITA and inactivating CD47) and then they are differentiated in a cell type for permanent transplantation in individuals. Thus, for example, HIP cells can be differentiated into hepatocytes (dHIP hepatocytes), pancreatic beta cells or islet organoids (beta dHIP cells), endothelial cells (dHIP endothelial cells), etc.
[0110] The term percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or
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41/105 subsequences that have a specified percentage of nucleotide or amino acid residues that are equal, when compared and aligned for maximum match, measured using one of the sequence comparison algorithms described below (for example, BLASTP and BLASTN or other algorithms available for verses technique) or by visual inspection. Depending on the application, the percentage of identity may exist over a region of the sequence to be compared, for example, over a functional domain, or, alternatively, it exists along the entire length of the two sequences to be compared. For sequence comparison, typically a sequence acts as a reference sequence in which tests are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the parameters of the sequence algorithm program are designated. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence (or sequences) relative to the reference sequence, based on the designated program parameters.
[0111] The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), using the similarity search method by Pearson & Lipman, Proc. Nat'1. Acad. Sci. USA 85: 2444 (1988), for computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.,
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Madison, Wis.), Or by visual inspection (see generally Ausubel et al., Infra).
[0112] An example of an algorithm that is stable for determining percentage of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. , J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
[0113] Inhibitors, activators and modulators affect a function or expression of a biologically relevant molecule. The modulator term includes inhibitors and activators. They can be identified using in vitro and in vivo assays for expression or activity of a target molecule.
[0114] Inhibitors are agents that, for example, inhibit expression or bind to target molecules- or proteins. They may partially or totally block stimulation or have protease inhibitory activity. They can reduce, decrease, prevent or delay activation, including inactivation, desensitization or negative regulation of the described target protein activity. Modulators can be antagonists of the target molecule- or protein.
[0115] Activators are agents that, for example, induce or activate the function or expression of a target molecule- or protein. They can bind, stimulate, increase, open, activate or facilitate the activity of the target molecule. Activators can be agonists of the target molecule- or protein.
[0116] Homologs are bioactive molecules that are similar to a reference molecule in the nucleotide sequence, peptide sequence, functional level or
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43/105 structural. Homologues can include derivatives of sequences that share a certain percentage of identity with the reference sequence. Thus, in one embodiment, homologous or derived sequences share at least 70 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least 80 or 85 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least 90 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least 95 percent sequence identity. In a more specific embodiment, homologous or derived sequences share at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99 percent sequence identity. Homologous or derivative nucleic acid sequences can also be defined by their ability to remain linked to a nucleic acid sequence under stringent hybridization conditions. Homologues with structural or functional similarity to a reference molecule can be chemical derivatives of the reference molecule. Methods for detecting, generating and scanning structural and functional homologs as well as derivatives are known in the art.
[0117] Hybridization generally depends on the ability of denatured DNA to revive when complementary strands are present in an environment below its fusion temperature. The greater the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is that higher relative temperatures tend to make the reaction conditions
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44/105 more stringent, while lower temperatures are less stringent. For further details and explanations of the rigor of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), hereby incorporated by reference in their entirety.
[0118] The rigor of the hybridization reactions is easily determined by one skilled in the art and generally an empirical calculation depending on the length of the probe, the washing temperature and the salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.
[0119] Strict conditions or conditions of high rigor, as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 sodium citrate 0.1% M / Dedecyl sulfate at 50 ° C; (2) during the hybridization, use a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / sodium phosphate buffer 50 Mm at Ph 6.5 with sodium chloride 750 Mm, sodium citrate 75 Mm at 42 ° C; or (3) overnight hybridization in a solution using 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 Mm sodium phosphate (Ph 6.8 ), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μΐ / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, with a wash for 10 minutes at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) followed by
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45/105 minutes thorough washing consisting of 0.1 x SSC containing EDTA at 55 ° C.
[0120] It is intended that each maximum numerical limitation given throughout this specification includes all the lower numerical limitations, as if such lower numerical limitations were expressly written here. Each minimum numerical limitation given throughout this specification will include all the highest numerical limitations, as if such higher numerical limitations were expressly written here. Each numerical range given throughout this specification will include all the narrowest numerical ranges that fall within such a large numerical range, as if such narrower numerical ranges were all expressly written here.
[0121] As used herein, the term modification refers to an alteration that physically differentiates the modified molecule from the parent molecule. In one embodiment, an amino acid change in a CD47, HSVtk, EC-CD or iCasp9 variant polypeptide prepared according to the methods described here differentiates it from the corresponding parent that has not been modified according to the methods described here, such as proteins wild-type, a naturally occurring mutant protein or other modified protein that does not include the modifications of such a variant polypeptide. In another embodiment, a variant polypeptide includes one or more modifications that differentiate the function of the variant polypeptide from the unmodified polypeptide. For example, an amino acid change in a variant polypeptide affects its receptor binding profile. In other embodiments, a variant polypeptide comprises substitution, deletion or
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46/105 insertion, or combinations thereof. In another embodiment, a variant polypeptide includes one or more modifications that increase its affinity for a receptor compared to the affinity of the unmodified polypeptide.
[0122] In one embodiment, a variant polypeptide includes one or more substitutions, insertions or deletions with respect to a corresponding native or parental sequence. In certain embodiments, a polypeptide
variant includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41 to 50 or 51 or more modifications. [0123] Per ' epissomal vector understands up on here one
genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; for example, it is not integrated into the host cell's genomic DNA. Various episomal vectors are known in the art and described below.
[0124] By knockout in the context of a gene means that the host cell that houses the knockout does not produce a functional protein product of the gene. As presented here, a knockout can result in a variety of ways, removing all or part of the coding sequence, introducing frame shift mutations so that a functional protein is not produced (truncated or meaningless sequence), removing or changing a component regulator (for example, a promoter) so that the gene is not transcribed, preventing translation through binding to mRNA, etc. The knockout is usually carried out at the level of genomic DNA, so that the descendants of the cells also carry the knockout permanently.
[0125] By knockin in the context of a gene
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47/105 means that the host cell that houses the knockin has more functional protein active in the cell. As presented here, a knockin can be performed in several ways, usually by introducing at least one copy of a transgene (tg) that encodes the protein into the cell, although this can also be done by replacing regulatory components as well, for example, by adding a constitutive promoter to the endogenous gene. In general, interference technologies result in the integration of the extra copy of the transgene into the host cell.
VII. CELLS OF THE INVENTION [0126] The invention provides compositions and methodologies for generating HIP cells, starting with wild-type cells, making the same pluripotent (e.g., producing induced pluripotent stem cells, or iPSCs), generating HIP cells from the iPSC population.
A. METHODOLOGIES FOR GENETIC CHANGES [0127] The invention includes methods of modifying nucleic acid sequences within cells or in cell-free conditions to generate both pluripotent cells and HIP cells. Exemplary technologies include homologous recombination, knockin, ZFNs (zinc finger nucleases), TALENs (effector nucleases similar to transcription activators), CRISPR (short palindromic repetitions grouped regularly, interspace) / Cas9 and other site-specific nuclease technologies. These techniques allow double-stranded DNA breaks at the desired locus sites. These controlled double-strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of
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48/105 nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce a double strand break in the nucleic acid molecule. The double-strand break is repaired by a non-homologous error-prone junction (NHEJ) or by homologous recombination (HR).
[0128] As will be appreciated by those skilled in the art, several different techniques can be used to manipulate the pluripotent cells of the invention, as well as the manipulation of iPSCs to become hypoimmunogenic, as presented herein.
[0129] In general, these techniques can be used individually or in combination. For example, in the generation of HIP cells, CRISPR can be used to reduce the expression of B2M and / or CIITA protein active in the manipulated cells, with viral techniques (for example, lentivirus) to perform CD47 functionality knockin. Also, as will be appreciated by those skilled in the art, although a modality sequentially uses a CRISPR step to perform B2M knockout, followed by a CRISPR step to perform CIITA knockin with a final step of a lentivirus to perform knockin on CD47 functionality, these genes can be handled in different orders using different technologies.
[0130] As discussed in more detail below, transient expression of reprogramming genes is generally performed to generate / induce pluripotent stem cells.
A. CRISPR TECHNOLOGIES [0131] In one embodiment, cells are
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49/105 manipulated with the use of short palindromic repetition technologies with regularly spaced clusters) / Cas (CRISPR) as is known in the art. CRISPR can be used to generate the starting iPSCs or to generate the HIP cells from the iPSCs. There are a large number of techniques based on CRISPR, see, for example Doudna and Charpentier, Science doi: 10.1126 / science.1258096, incorporated here for reference. CRISPR techniques and kits are sold commercially.
B. TALEN TECHNOLOGIES [0132] In some embodiments, the HIP cells of the invention are produced using Transcription Activator-type Effector Nucleases (TALEN) methodologies. TALEN are restriction enzymes combined with a nuclease that can be modified to bind and cut almost any desired DNA sequence. TALEN kits are sold commercially.
C. ZINC FINGER TECHNOLOGIES [0133] In one embodiment, cells are manipulated using Zn finger nuclease technologies. The nucleases of the Zn finger are artificial restriction enzymes generated by the fusion of a DNA binding domain of the zinc finger to a DNA dividing domain. Zinc finger domains can be manipulated to achieve specific desired DNA sequences and this allows zinc finger nucleases to target unique sequences within complex genomes. Leveraging endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALENs.
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D. VIRUS-BASED TECHNOLOGIES [0134] There is a wide variety of viral techniques that can be used to generate the HIP cells of the invention (as well as for the original generation of iPCSs), including, without limitation, the use of retroviral vectors, vectors lentivirals, adenovirus vectors and Sendai viral vectors. The episomal vectors used in the generation of iPSCs are described below.
E. DESCENDING REGULATION OF GENES USING INTERFERENT RNA [0135] In other embodiments, genes that encode proteins used in HLA molecules are downregulated by RNAi technologies. RNA interference (RNAi) is a process in which RNA molecules inhibit gene expression, usually causing the degradation of specific mRNA molecules. Two types of RNA molecules - microRNA (miRNA) and small interfering RNA (siRNA) - are central to RNA interference. They bind to the target mRNA molecules and increase or decrease their activity. RNAi helps cells defend against parasitic nucleic acids, such as those from viruses and transposons. RNAi also influences development.
[0136] sdRNA molecules are a class of asymmetric siRNAs comprising a 19-21 base guide (antisense) chain. They contain pyrimidines modified with 5 'phosphate, 2'Ome or 2'F and six phosphotioates in the 3' positions. They also contain a sensitive chain containing conjugated 3 'portions of sterol, 2 phosphoioates at the 3' position and 2'0me modified pyrimidines. Both chains contain 2'Ome purines with continuous stretches of unmodified purines that are not
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51/105 exceeds a length of 3. sdRNA is disclosed in US Patent No. 8,796,443, hereby incorporated by reference in its entirety.
[0137] For all of these technologies, well-known recombinant techniques are used to generate recombinant nucleic acids, as presented herein. In certain embodiments, recombinant nucleic acids (which can encode a desired polypeptide, for example, CD47, or disruption sequences) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell and subject to treatment. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, one or more regulatory nucleotide sequences may include, without limitation, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription start and stop sequences, translation start and stop sequences and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. Promoters can be naturally occurring promoters or hybrid promoters that combine elements from more than one promoter. An expression construct can be present in a cell in an episome, such as a plasmid, or an expression construct can be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain modalities include an expression vector comprising a
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52/105 nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. The regulatory sequence for use here includes promoters, enhancers and other elements of expression control. In certain embodiments, an expression vector is designed for choosing the host cell to be transformed, the specific variant polypeptide to be expressed, the number of copies of the vector, the ability to control that number of copies or the expression of any other protein encoded by the vector, as antibiotic markers.
[0138] Examples of promoters in mammal suitable include, for example, promoters From next genes: promoter of ubiquitin / S27a hamster (WO 97/15664),
simian vaccine vacuolation virus (SV40) early promoter, adenovirus major late promoter, mouse metallothionein I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), the promoter of the mammary tumor virus of mouse (MMTV), the long terminal repeat region of the Moloney murine leukemia virus and the early promoter of human cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoter (or promoters).
[0139] In additional embodiments, promoters for use in mammalian host cells can be obtained from virus genomes, such as polyomavirus virus, chicken poxvirus (UK 2211504 published July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40). In other embodiments, heterologous mammalian promoters are used. Examples include the actin promoter,
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53/105 an immunoglobulin promoter and heat shock promoters. SV40 early and late promoters are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. Fiers et al. , Nature 273: 113-120 (1978). The immediate initial promoter of human cytomegalovirus is conveniently obtained as a HindIIIE restriction fragment. Greenaway, PJ et al. , Gene 18: 355-360 (1982). Previous references are incorporated by reference in their entirety.
B. PLURIPOTENT CELL GENERATION [0140] The invention provides methods of producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to supply pluripotent stem cells.
[0141] The generation of pluripotent mouse and human stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those skilled in the art, there are a variety of different methods for generating iPCSs. The original induction was performed from embryonic or adult fibroblasts from mice using the viral introduction of four transcription factors, Oct3 / 4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126: 663-676 (2006), incorporated herein by reference in its entirety and specifically for the techniques outlined therein. Since then, several methods have been developed; see Seki et al., World J. Stem Cells 7 (1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, which are here
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54/105 expressly incorporated by reference in their entirety, and in particular for hiPSC generation methods (see, for example, Chapter 3 of that latter reference).
[0142] Generally, iPSCs are generated by the transient expression of one or more reprogramming factors in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of cells are induced to become iPSCs (in general, the efficiency of this step is low, since no selection marker is used). Once the cells are reprogrammed, and become pluripotent, they lose the episomal vector and produce factors using endogenous genes. This loss of the episomal vector (or vectors) results in cells called zero footprint cells. This is desirable, as the less genetic modifications (particularly in the host cell genome), the better. Thus, it is preferable that the resulting hiPSCs do not have permanent genetic modifications.
[0143] As is also appreciated by those skilled in the art, the number of reprogramming factors that can be used or are used may vary. Commonly, when fewer reprogramming factors are used, the efficiency of transforming cells into a pluripotent state decreases, as well as pluripotency, for example, less reprogramming factors can result in cells that are not fully pluripotent, but may only have the capacity to differentiate into fewer cell types.
[0144] In some modalities, a single reprogramming factor, OCT4, is used. In other modalities, two reprogramming factors, OCT4 and KLF4, are used. In other modalities, three risk factors are used
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55/105 reprogramming, OCT4, KLF4 and SOX2. In other modalities, four reprogramming factors are used, OCT4, KLF4, SOX2 and c-Myc. In other modalities, 5, 6 or 7 reprogramming factors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28 and SV40L T antigens.
[0145] In general, these reprogramming factor genes are supplied in episomal vectors as they are known in the art and are commercially available. For example, ThermoFisher / Invitrogen sells a sendai virus reprogramming kit for generation of zero footprint hiPSCs, see catalog number A34546. ThermoFisher also sells EBNA-based systems, see catalog number A14703.
[0146] In addition, there are a number of commercially available hiPSC lines; see, for example, Gibco® HiPSC Epissomal line, K18945, which is a human iPSC cell line free of zero footprint viral integration (see also Burridge et al., 2011, supra).
[0147] In general, as is known in the art, iPSCs are produced from non-pluripotent cells, such as CD34 + cord blood cells, fibroblasts, etc., transiently expressing the reprogramming factors as described herein.
[0148] For example, successful iPSCs were also generated using only Oct3 / 4, Sox2 and Klf4, while omitting C-Myc, although with reduced reprogramming efficiency.
[0149] In general, iPSCs are characterized by the expression of certain factors including KLF4, Nanog, OCT4,
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SOX2, ESRRB, TBX3, c-Myc and TCL1. The new or increased expression of these factors for the purposes of the invention can be via induction or modulation of an endogenous locus or from expression from a transgene.
[0150] For example, murine iPSCs can be generated using the methods of Diecke et al., Sei Rep. 2015, January 28; 5: 8081 (doi: 10.1038 / srep08081), incorporated herein by reference in its entirety and specifically for the methods and reagents for the generation of miPSCs. See also, for example, Burridge et al. , PLoS One, 2011 6 (4): 18293, hereby incorporated by reference in its entirety and specifically for the methods presented here.
[0151] In some cases, cell pluripotency is measured or confirmed as shown here, for example, by testing reprogramming factors, as is generally shown in Figure 17, or by performing differentiation reactions, as outlined here and in the Examples.
C. GENERATION OF HYPOIMMUNOGENIC PLURIPOTENT CELLS [0152] The present invention is directed to the generation, manipulation, growth and transplantation of hypoimmunogenic cells in a patient as defined here. The generation of HIP cells from pluripotent cells is carried out with only three genetic alterations, resulting in the minimum disruption of cellular activity, but conferring immunosilencing to the cells.
[0153] As discussed here, one modality uses a reduction or elimination in the protein activity of MHC I and II (HLA I and II when the cells are human). This can be done by changing the genes that encode your
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57/105 components. In one embodiment, the coding region or regulatory sequences of the gene are disrupted with the use of CRISPR. In another modality, gene translation is reduced with the use of interfering RNA technologies. The third change is a change in a gene that regulates macrophage susceptibility to phagocytosis, such as CD47, and this is usually a knockin of a gene using viral technologies.
[0154] In some cases, where CRISPR is being used for genetic modifications, hiPSC cells that contain a Cas9 construct that allows high-efficiency editing of the cell line can be used; see, for example, the iPSC Cas9 Epissomal Human cell line, A33124, from Life Technologies.
1. REDUCTION OF HLA-I [0155] The HIP cells of the invention include a reduction in the function of MHC I (HLA I when the cells are derived from human cells).
[0156] As will be appreciated by those skilled in the art, reduction of function can be achieved in several ways, including removing nucleic acid sequences from a gene, interrupting the sequence with other sequences or changing the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced by meaningless sequences, structure-changing mutations can be made, all or part of a regulatory sequence, such as a promoter can be removed or replaced, initiation strings can be removed or replaced, etc.
[0157] As will be understood by those skilled in the art, the successful reduction of MHC I function (HLA I
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58/105 when cells are derived from human cells) in pluripotent cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind to the HLA complex; for example, with the use of commercially available HLA-A, B, C antibodies that bind to the alpha chain of the major human histocompatibility class HLA antigens.
The. B2M CHANGE [0158] In one embodiment, the reduction in HLA-I activity is accomplished by disrupting the expression of the 2-2 microglobulin gene in the pluripotent stem cell, the human sequence of which is revealed here. This change is generally referred to here as a gene knockout, and in the HIP cells of the invention it is carried out on both alleles in the host cell. Generally, the techniques for making the two breaks are the same.
[0159] A particularly useful modality uses CRISPR technology to disrupt the gene. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of the gene, so that no functional proteins are produced, often the result of frame shift mutations that result in the generation of mode stop codons. that truncated, non-functional proteins are produced.
[0160] Consequently, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mice or the B2M gene in humans. After genetic editing, the transfected iPSC cultures are dissociated into individual cells. At
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59/105 individual cells are expanded to full-size colonies and tested for CRISPR editing by scanning for the presence of aberrant sequences from the CRISPR dividing site. Clones with deletions in both alleles are chosen. Such clones did not express B2M / B2M as demonstrated by PCR and did not express HLA-I as demonstrated by FACS analysis (see examples 1 and 6, for example).
[0161] Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of used cells probed with antibodies to the B2M protein. In another embodiment, polymerase chain reactions with reverse transcriptase (rt-PCR) confirm the presence of the inactivating alteration.
[0162] In addition, cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This can be assayed by FACS analysis using antibodies to one or more components of the HLA cell surface, as discussed above.
[0163] It is noteworthy that others had poor results when trying to silence the B2M genes in both alleles. See, for example, Gornalusse et al. , Nature Biotech .Doi / 10. 1038 / nbt. 3860).
[0164] In addition to a reduction in HLA I, the HIP cells of the invention also lack MHC II function (HLA II when the cells are derived from human cells).
[0165] As will be appreciated by those skilled in the art, reduction of function can be achieved in several ways, including removing nucleic acid sequences from a gene, adding nucleic acid sequences to a gene, disrupting the reading frame, interrupting the sequence
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60/105 with other sequences, or by changing the regulatory components of nucleic acid. In one embodiment, all or part of a coding region of the gene of interest can be removed or replaced with meaningless sequences. In another embodiment, regulatory sequences, such as a promoter, can be removed or replaced, translation initiation sequences can be removed or replaced, etc.
[0166] Successful reduction of MHC II function (HLA II when cells are derived from human cells) in pluripotent cells or their derivatives can be measured using techniques known in the art like Western blotting using antibodies to the protein, FACS techniques , rt-PCR, etc.
The. CIITA CHANGE [0167] In one embodiment, the reduction of HLA-II activity is accomplished by disrupting the expression of the CIITA gene in the pluripotent stem cell, the human sequence of which is illustrated here. This change is generally referred to here as a gene knockout, and in the HIP cells of the invention it is carried out on both alleles in the host cell.
[0168] Assays to test whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies to the CIITA protein. In another embodiment, polymerase chain reactions with reverse transcriptase (rtPCR) confirm the presence of the inactivating alteration.
[0169] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this test is performed as is known in the art (see Figure 21, for example) and
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61/105 is usually performed using Western Blots or FACS analysis based on commercial antibodies that bind to human HLA-II HLA-DR, DP antigens and most DQ antigens as described below.
[0170] A particularly useful modality uses CRISPR technology to disrupt the CIITA gene. CRISPRs were designed to target the coding sequence of the Ciita gene in the mouse or the CIITA gene in humans, an essential transcription factor for all MHC II molecules. After genetic editing, the transfected iPSC cultures were dissociated into single cells. They were expanded to full-size colonies and tested for successful CRISPR editing through screening for the presence of an aberrant sequence from the CRISPR dividing site. Deletion clones do not express Ciita / CIITA as determined by POR and do not express MHC II / HLA-II as determined by FACS analysis.
3. Phagocytosis reduction [0171] In addition to reduction of HLA I and II (or MHC I and II), usually with the use of B2M and CIITA knockouts, the HIP cells of the invention have a reduced susceptibility to macrophage phagocytosis and death of NK cells. The resulting HIP cells escape the immune macrophage and innate pathways due to one or more CD47 transgenics.
A. INCREASE IN CD47 [0172] In some embodiments, reduced phagocytosis of macrophages and susceptibility to death of NK cells result from the increase in CD47 on the HIP cell surface. This is accomplished in several ways, as will be appreciated by those skilled in the art using knockin or transgenic technologies. In some cases, increased expression of CD47
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62/105 results from one or more CD47 transgene.
[0173] Consequently, in some embodiments, one or more copies of a CD47 gene are added to HIP cells under the control of an inducible or constitutive promoter, where the latter is preferred. In some embodiments, a lentiviral construct is employed as described or known in the art. CD47 genes can integrate into the host cell genome under the control of a suitable promoter as is known in the art.
[0174] The HIP cell lines were generated from B2M - / - CIITA - / - iPSCs. Cells containing lentivirus vectors expressing CD47 were selected using a Blasticidin marker. The CD47 gene sequence was synthesized and the DNA was cloned into the plasmid Lentivirus pLenti6 / V5 with a resistance to blasticidin (Thermo Fisher Scientific, Waltham, MA, USA).
[0175] In some embodiments, the expression of the CD47 gene can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by exchanging the endogenous promoter for a constitutive promoter or for a different inducible promoter. This can usually be accomplished using known techniques, such as CRISPR.
[0176] Once altered, the presence of sufficient CD47 expression can be assayed using techniques known as those described in the Examples, such as Western blots, ELISA assays or FACS assays using anti-CD47 antibodies. In general, sufficiency in this context means an increase in the expression of CD47 on the surface of the HIP cell that silences the death of NK cells. The levels of natural expression in cells are very low to protect
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63/105 the same against the Use of NK cells, once their MHC I is removed.
4. SUICIDAL GENES [0177] In some embodiments, the invention provides hypoimmunogenic pluripotent cells that comprise a suicide or suicide switch gene. They are incorporated to function as a safety switch that can cause the death of hypoimmunogenic pluripotent cells, if they grow and divide unwantedly. The suicide gene ablation approach includes a suicide gene in a gene transfer vector that encodes a protein that results in cell death only when activated by a specific compound. A suicide gene can encode an enzyme that selectively converts a non-toxic compound to highly toxic metabolites. The result is specifically to eliminate the cells that express the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir. In other modalities, the suicide gene is the Escherichia coli cytosine deaminase gene (EC-CD) and the stimulus is 5 fluorocytosine (5-FC) (Barese et al., Mol. Therap 20 (10): 1932-1943 ( 2012), Xu et al., Cell Res. 8: 73-8 (1998), both of which are incorporated herein by reference in their entirety).
[0178] In other embodiments, the suicide gene is an inducible Caspase protein. An inducible caspase protein comprises at least a portion of a Caspase protein capable of inducing apoptosis. In one embodiment, the portion of the Caspase protein is exemplified in SEQ ID NO: 6. In preferred embodiments, the inducible Caspase protein is iCasp9. It comprises the sequence of the binding protein
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64/105 to human FK506, FKBP12, with an F36V mutation, linked through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small molecule dimerizing agent, AP1903. Thus, the suicidal function of iCasp9 in the present invention is triggered by the administration of a chemical dimerization inducer (CID). In some embodiments, the CID is the AP1903 small molecule drug. Dimerization causes rapid induction of apoptosis. (See WO2011146862; Stasi et al, N. Engl. J. Med 365; 18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13: 913-924 (2007), each is incorporated herein by reference in its entirety).
5. TESTING FOR HIP PHENOTYPES AND RETENTION OF PLURIPOTENTIALITY [0179] Once HIP cells have been generated, they can be analyzed for their hypoimmunogenicity and / or retention of pluripotency, as is generally described here and in the examples.
[0180] For example, hypoimmunogenicity is assayed using various techniques as exemplified in Figure 13 and Figure 15. These techniques include transplantation into allogeneic hosts and monitoring the growth of HIP cells (eg, teratomas) that escape the immune system host. HIP derivatives are transduced to express luciferase and can be followed using bioluminescence images. Similarly, the response of the host animal's T cells and / or B cells to the HIP cells is tested to confirm that the HIP cells do not cause an immune reaction in the host animal. T cell function is assessed by Elispot, Elisa, FACS, PCR or mass cytometry (CYTOF). THE
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65/105 B cell response or antibody response is assessed using FACS or luminex. Additionally or alternatively, cells can be tested for their ability to prevent innate immune responses, for example, death of NK cells, as is generally shown in Figure 14. The lipolytic activity of NK cells assessed in vitro or in vivo (as shown in Figure 15).
[0181] Of the same form, The retention pluripotency is tested on several ways. In a modality, pluripotency is rehearsed by expression in certain factors
specific pluripotency, as generally described herein and shown in Figure 29. In addition or alternatively, HIP cells are differentiated into one or more cell types as an indication of pluripotency.
d. PREFERENTIAL MODALITIES OF THE INVENTION [0182] Hypoimmunogenic pluripotent stem cells (HIP cells) are provided here that exhibit pluripotency but do not result in a host immune response when transplanted into an allogeneic host, as a human patient, either as HIP cells or as products differentiated from HIP cells.
[0183] In one embodiment, human pluripotent stem cells (hiPSCs) are made hypoimmunogenic by a) the disruption of the B2M gene in each allele (eg B2M / -), b) the disruption of the CIITA gene in each allele ( for example, CIITA - / -) and c) by overexpressing the CD47 gene (CD47 +, for example, introducing one or more additional copies of the CD47 gene or activating the genomic gene). This renders the hiPSC B2M - / - CIITA - / - CD47tg population. In a preferred embodiment, the cells are not immunogenic. In another
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66/105 modality, the HIP cells are rendered non-immunogenic B2MCIITAas described above, but are further modified by inclusion of a suicide gene induced which is induced to kill the cells in vivo when necessary.
and. MAINTENANCE OF HIP CELLS [0184] Once generated, HIP cells can be maintained in an undifferentiated state, as is known for the maintenance of iPSCs. For example, HIP cells are grown in Matrigel using culture medium which prevents differentiation and maintains pluripotency.
f. HIP CELL DIFFERENTIATION [0185] The invention provides HIP cells that are differentiated into different cell types for subsequent transplantation in individuals. As will be appreciated by those skilled in the art, methods for differentiation depend on the type of cell desired with the use of known techniques. The cells are differentiated into suspension and then placed in a gel matrix form, such as matrigel, gelatin or fibrin / thrombin forms to facilitate cell survival. Differentiation is assayed as is known in the art, usually by assessing the presence of specific cell markers.
[0186] In some embodiments, HIP cells are differentiated into hepatocytes to address the loss of hepatocyte function or cirrhosis of the liver. There are several techniques that can be used to differentiate HIP cells into hepatocytes; see for example Pettinato et al., doi: 10.1038 / spre32888, Snykers et al. , Methods Mol Biol 698: 305-314 (2011), Si-Tayeb et al. , Hepatology 51: 297-305 (2010) and Asgari et al. , Stem Cell Rev (: 493-504 (2013), all
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67/105 which are expressly incorporated herein by reference in their entirety and specifically for methodologies and reagents for differentiation. Differentiation is assayed as is known in the art, usually by assessing the presence of associated and / or specific hepatocyte markers, including, without limitation, albumin, alpha-fetoprotein and fibrinogen. Differentiation can also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release and glycogen storage.
[0187] In some embodiments, HIP cells are differentiated into cells similar to beta or islet organoids for transplantation to treat type I diabetes mellitus (DM1). Cellular systems are a promising way to approach DM1, see, eg, Ellis et al., Doi / 10.1038 / nrgastro. 2017.93, incorporated herein by reference. In addition, Pagliuca et al. reports the successful differentiation of β cells from hiPSCs (see doi / 10.106 / j. cell. 2014.09.040, hereby incorporated by reference in its entirety and in particular for the methods and reagents presented here for large-scale cell production human β from human pluripotent stem cells). In addition, Vegas et al. shows the production of human β cells from human pluripotent stem cells, followed by encapsulation to prevent immune rejection by the host; (doi: 10.1038 / nm. 4030, incorporated herein by reference in its entirety and in particular for the methods and reagents presented there for the large-scale production of functional human cells from human pluripotent stem cells).
[0188] Differentiation is tested as it is
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68/105 known in the art, generally assessing the presence of associated or specific β cell markers, including without insulin limitation. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, doi: 10.1016 / j. cell. 2016.09.002, incorporated herein by reference in its entirety, and specifically for the biomarkers presented here.
[0189] Once dHIP beta cells are generated, they can be transplanted (as a cell suspension or into a gel matrix as discussed here) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, a muscle or subcutaneous pockets.
[0190] In some modalities, HIP cells are differentiated into retinal pigment epithelium (RPE) to address diseases that threaten the vision of the eye. Human pluripotent stem cells were differentiated into RPE cells, using the techniques outlined in Kamao et al., Stem Cell Reports 2014: 2: 205-18, incorporated herein by reference in their entirety and in particular for methods and reagents presented there for differentiation techniques and reagents; see also Mandai et al. , doi: 10.1056 / NEJMoal608368, also incorporated in its entirety for techniques of generation of slides from EPR cells and transplantation in patients.
[0191] Differentiation can be tested as is known in the art, usually by assessing the presence of PSE-associated and / or specific markers or by measuring functionally. See, for example, Kamao et al., Doi: 10.1016 / j. sterner.2013.12.007, incorporated herein by reference in its entirety and specifically for the
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69/105 markers presented in the first paragraph of the results section.
[0192] In some embodiments, HIP cells are differentiated into cardiomyocytes to treat cardiovascular disease. Techniques are known in the art for differentiating hiPSCs from cardiomyocytes and discussed in the Examples. Differentiation can be tested as is known in the art, usually by assessing the presence of associated or specific cardiomyocyte markers or by measuring functionally; see for example Loh et al. , doi: 10.1016 / j. cell.2016.06.001, hereby incorporated by reference in its entirety and specifically for methods of differentiating stem cells including cardiomyocytes.
[0193] In some embodiments, HIP cells are differentiated into endothelial colony-forming cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., Doi: 10.1038 / nbt.3048, incorporated by reference in its entirety and specifically for methods and reagents for the generation of endothelial cells from human pluripotent stem cells, and also for techniques transplant. Differentiation can be tested as is known in the art, usually by assessing the presence of associated or specific markers of the endothelial cell or by measuring functionally.
[0194] In some embodiments, HIP cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones to address autoimmune thyroiditis. Techniques to differentiate
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70/105 thyroid cells are known in the art. See, for example, Kurmann et al. , doi: 10,106 / j. stem.2015.09.004, here expressly incorporated by reference in its entirety and specifically for the methods and reagents for the generation of thyroid cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be tested as is known in the art, usually by assessing the presence of associated or specific markers of thyroid cells or by measuring functionally.
g. DIFFERENTIATED HIP CELL TRANSPLANTATION [0195] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on both the type of cell and the end use of these cells. In general, the dHIP cells of the invention are transplanted intravenously or by injection in particular locations of the patient. When transplanted in specific locations, cells can be suspended in a gel matrix to prevent dispersion while they are fixed.
[0196] In order for the invention described here to be more fully understood, the following examples are presented. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any way.
viii. EXAMPLES
The. GENERAL TECHNIQUES
1. GENERATION OF MICE IPSCs [0197] These cells were generated using the methods of Diecke et al, Scl Rep. 2015, 28 January; 5: 8081 (doi: 10.1038 / srep08081), incorporated herein in its entirety and
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71/105 specifically for the methods and reagents for the generation of miPSCs.
[0198] Mouse murine tail fibroblasts were dissociated and isolated with collagenase type IV (Life Technologies, Grand Island, NY, USA) and maintained with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) , L-glutamine, 4.5 g / l glucose, 100 U / ml penicillin and 100 pg / ml streptomycin at 37 ° C, 20% O2 and 5% CO2 in a humidified incubator. 1 χ 10 ⁶ murine fibroblasts were then reprogrammed using a new codon-optimized mini-intronic plasmid (co-MIP) (10-12 pm DNA) expressing the four reprogramming factors Oct4, KLF4, Sox2 and c-Myc using the Neon Transfection system. After transfection, fibroblasts were plated in a MEE feeder layer and maintained in fibroblast media with the addition of sodium butyrate (0.2 mM) and 50 pg of ascorbic acid / ml. When the colonies appeared as CES-, the medium was switched to murine iPSC medium containing DMEM, 20% FBS, L-glutamine, non-essential amino acids (NEAA), β mercaptoethanol, and 10 ng / ml leukemia inhibitor factor (LIE). After 2 passes, the murine iPSCs were transferred to 0.2% gelatin-coated plates and subsequently expanded. With each pass, the iPSCs were classified for the murine pluripotency marker SSEA-1, using the classification of magnetic activated cells (MACS).
2. GENERATION OF HUMAN IPSCs [0199] The generation of hiPSCs was performed as generally described in Burridge et al., PLoS One, 2011 6 (4): 18293, here incorporated by reference in its entirety and specifically for the methods described here.
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72/105 [0200] The Gibco® Human Episomal iPSC Line (Catalog No. A33124, Thermo Fisher Scientific) was derived from CD34 + cord blood using an EBNA-based episomal system of three plasmids, seven factors ( SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen). This iPSC line is considered a zero footprint, since there was no integration into the genome from the reprogramming event. It has been shown to be free of all reprogramming genes.
[0201] Gibco® Human Epissomal iPSC Line has a normal karyotype and an endogenous expression of pluripotent markers such as Oct4, Sox2 and Nanog (as shown by RT-PCR) and Oct4, SSEA4, TRA-1-60 and TRA- 1-81 (as shown by the ICG). Expression of the entire genome and analysis of epigenetic profiles have shown that this hiPSC spisome line is molecularly indistinguishable from human embryonic stem cell lines (Burridge et al., 2011). In targeted differentiation and teratoma analyzes, these hiPSCs maintained their differentiation potential for ectodermal, endodermal and mesodermal strains (Burridge et al., 2011). In addition, vascular, hematopoietic, neural and cardiac strains were derived with robust efficiencies (Burridge et al., 2011).
3. FACS ANALYSIS OF SURFACE MOLECULES
The. DETECTION OF HLA I HUMAN SURFACE MOLECULES [0202] Human IPSCs, iCMs and iECs were plated in 6-well plates and stimulated with 100 ng / ml human IFNg (Peprotech, Rocket Hill, NJ). The cells were collected and labeled with HLA-A, B, C antibody conjugated to APC (clone G46_2.6, category No. 562006, BD BioSciences, San Jose, CA, USA) or IgGl isotype control antibody
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73/105 conjugated to APC (clone MOPC-21, category No. 555751, BD BioSciences). The HLA-A, B, C antibody binds to the alpha chain of HLA Class I antigens of human major histocompatibility. Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
4. DETECTION OF HUMAN HLA II SURFACE MOLECULES [0203] Human IPSCs, iCMs and iECs were plated in 6-well plates and stimulated with 100 ng / ml human TNFα (Peprotech, Rocket Hill, NJ). The cells were collected and labeled with HLA-DR, DP, DQ labeled Alexa-flour647 (clone Tu3a, category No. 563591, BD BioSciences, San Jose, CA, USA) or Alexa-labeled IgG2a isotype control antibody -fluor647 (clone G155-178, category No. 557715, BD BioSciences). The HLA-DR, DP, DQ antibody binds to human HLA-DR antigens, HLA-DR, DP and most DQ antigens. Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
5. DETECTION OF HUMAN CD47 SURFACE MOLECULES [0204] Human IPSCs, iCMs and iECs were plated in 6-well plates and stimulated with 100 ng / ml human IFNg (Peprotech, Rocket Hill, NJ). The cells were collected and labeled with CD47 conjugated to PerCP-Cy5 (clone B6H12, category No. 561261, BD BioSciences, San Jose, CA, USA) or IgGl isotype control antibody conjugated to PerCP-Cy5 (clone MOPC-21 , category No. 550795, BD BioSciences). The monoclonal antibody B6H12 CD47 specifically binds to CD47, an N-linked glycan protein of
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42-52 kDa. Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
6. DETECTION OF MUSEUM SURFACE MOLECULES OF MICE I [0205] For the detection of MHC I surface molecules in miPSC, miEC, miSMC and miCM, cells were plated in 6-well plates coated with gelatin and stimulated with 100 ng / ml of mouse IFNg (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with PerCP-eFlour710-labeled MHCI antibody (clone AF688.5.5.3, cat. 46-5958-82, eBioscience, Santa Clara, CA, USA) or IgG2b isotype control antibody labeled PerCPeFlour710. (clone eB149 / 10H5, cat. 46-4031-80, eBioscience). The MHCI antibody reacts with the MHC class I H 2K alloantigen. Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
7. DETECTION OF MUSEUM MHC II SURFACE MOLECULES [0206] For the detection of MHC II surface molecules in miPSC, miEC, miSMC and miCM, cells were plated in 6 well plates coated with gelatin and stimulated with 100 ng / ml mouse TNFα (Peprotech, Rocket Hill, NJ). After collection, the cells were stained with MCP II antibody labeled with PerCP-eFlour710 (clone M5 / 114.15.2, cat. 46-5321-82, eBioscience, Santa Clara, CA) or control antibody of the labeled IgG2a / K isotype with PerCPeFlour710. (clone eBM2a, cat. 46-4724-80, eBioscience). HC MHC II antibody reacts with the
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75/105 major histocompatibility of class II mice, both glycoproteins encoded in the ΙΑ and IE subregion. Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
8. DETECTION OF MOUSE CD47 SURFACE MOLECULES [0207] For the detection of Cd47 surface molecules in miPSC, miEC, miSMC and miCM, cells were plated on 6 well plates coated with gelatin and stimulated with mouse IFNg at 100 ng / ml (Peprotech, Rocket Hill, NJ, USA). After collection, cells were labeled with Alexa Fluor 647-labeled Cd47 antibody (miapSOl clone, category No. 563584, BD BioSciences, San Jose, CA, USA) or Alexa Fluor-labeled IgG2a / K isotype control antibody 647. (clone R35-95, category No. 557690, BD BioSciences). The Cd47 antibody specifically binds to the extracellular domain of CD47, also known as Integrin Associated Protein (TAP). Data analysis was performed by flow cytometry (BD Bioscience) and the results were expressed as a fold change for isotype control.
9. DETERMINING CELLULAR MORPHOLOGY OF MICE IN VIVO AFTER ALLOGENIC TRANSPLANTATION [0208] Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 myelo cells, miPSC-derived cardiomyocytes (miCM), miPSC-derived smooth muscle cells (miSMC) or miPSC-derived endothelial cells (miEC) in 250 ui of 0.9% saline were mixed with 250 ui of BD Matrigel High Concentration (1: 1 BD Biosciences)
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76/105 and injected subcutaneously in the lower dorsal of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10 and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin inclusion. . A 5 pm thick section was cut and stained with Hematoxylin and Eosin (HE).
10. DETERMINATION OF HUMAN CELL MORPHOLOGY IN VIVO AFTER ALLOGENIC TRANSPLANTATION [0209] Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 medium of cells, cardiomyocytes derived from hiPSC (hiCM) or endothelial cells derived from hiPSC (hiEC) in 250 µl of 0.9% saline solution containing ZVAD (100 mM, benzyloxycarbonyl-ValAla-Asp (O-methyl) -fluoromethylketone , Calbiochem), Bcl-XL BH4 (TAT peptide permeated by cells, 50 nM, Calbiochem), cyclosporin A (200 nM, Sigma), IGF-1 (100 ng / ml, Peprotech) and pinacidil (50 mM, Sigma) were mixed with 250 ui of BD Matrigel High Concentration (1: 1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 2, 4, 6, 8, 10 and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin inclusion. A 5 pm thick section was cut and stained with Hematoxylin and Eosin (HE).
B. EXAMPLE 1: GENERATION OF β-2 MICROGLOBULIN KNOCKOUT PLURIPOTENT CELLS IN A MOUNTAIN MODEL [0210] Induced Pluripotent Cell Generation:
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77/105 hypoimmune pluripotent cells were generated in a mouse modality. Human hypoimmune pluripotent cells are another modality that are generated using the strategies described here.
[0211] Mouse-induced pluripotent stem cells (miPSCs) were generated from C57BL / 6 fibroblasts. Mitomycin-inhibited CF1 mouse embryonic fibroblasts (MEF, Applied Stemcell, CA) were thawed and maintained in DMEM + GlutaMax 31966 (Gibco, Grand Island, NY, USA) with 10% heat inactivated fetal calf serum (FCS hi) , MEM-NEAA 1% and Pen Strep 1% (Thermo Fisher Scientific Gibco, Waltham, MA, USA). After MEF feeder cells formed a 100% confluent monolayer, miPSCs were cultured in MEF in KO DMEM 10829 with 15% KO serum replacement, 1% MEM-NEAA, 1% Pen Strep (Thermo Fisher-Gibco), 1x beta-mercaptoethanol and 100 LIE units (Millipore, Billerica, MA, USA). The cells were kept in 10 cm plates, the medium was changed daily and the cells were passed every 2-3 days with the use of 0.05% Trypsin-EDTA (Thermo Fisher-Gibco). The miPSCs were grown on gelatin (Millipore) without feeders using conventional means. Cell cultures were regularly analyzed for mycoplasma infections using the MycoAlert Kit (Lonza, Cologne, Germany).
[0212] Mice: BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU, CAnN. CgFoxnl // Cri, H2d) and light beige (CBySmn. CB17-Prkdcscid / J) (all 6-12 weeks) were used as recipients for different assays (all 6-12 weeks old). The mice were purchased at Charles River Laboratories
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78/105 (Sulzfeld, Germany) and received humanitarian care according to the Guide for Principles of Laboratory Animals. Animal experiments were approved by the Amt fur Gesundheit und Verbraucherschutz in Hamburg and carried out in accordance with EU and local guidelines.
[0213] Pluripotency Confirmation: Pluripotency has been demonstrated by rtPCR. The RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific). A DNase I step was included to remove the contaminating genomic DNA. The cDNA was generated using Applied Biosystems ® high capacity cDNA reverse transcription kit. Controls without reverse transcriptase (without RT) were also generated from all RNA samples. Specific gene primers were used to amplify target sequences using the AmpliTaq Gold 360 Master Mix (Thermo Fischer Fisher-Applied Biosystems, Waltham, MA, USA). The PCR reactions were visualized on 2% agarose gels. A set of positive control primers has been included that amplifies a constitutively expressed home maintenance gene (Actb) that encodes a cell cytoskeleton protein. The results are shown in Figure 2. Pluripotency markers Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcll were detected by rt-PCR of miPSC cells, but not by parental fibroblasts.
[0214] Pluripotency has also been tested by immunofluorescence. The miPSC were plated in 24-well plates and processed for analysis by RT-PCR and immunocytochemistry (ICC) 48 h after plating. For the ICC, the cells were fixed, permeabilized and blocked using the Image-iT Fixation / Permeabilization Kit (Thermo Fisher Scientific,
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Waltham, MA, USA). The cells were stained overnight at 4 ° C with primary antibodies to Sox2 and Oct4. After several washes, the cells were incubated with a secondary AlexaFluor 488 antibody and the NucBlue Fixed Cell ReadyProbes reagent (Thermo Fisher Scientific). The stained cells were visualized using a fluorescent microscope and were positive for Sox2 and Oct4. Data not shown.
[0215] Figure 3 shows additionally confirms pluripotency by a functional assay. 2 x 10 ⁶ miPSC cells were injected into the thigh muscle of C57BL / 6 recipient mice (syngenic), BALB / c mice (allogeneic), nude BALB / c mice (allogeneic, but not T cell deficient), and beige SCID mice (immunodeficient). Teratomas were formed in all mice, except immunocompetent allogeneic BALB / c mice.
[0216] β-2 Microglobulin Knockout: CRISPR technology was used for the knockout of the B2m gene. To target the coding sequence of the mouse β-2microglobulin gene (B2m), the CRISPR 5'TTCGGCTTCCCATTCTCCGG (TGG) -3 'sequence was paired and ligated into the All-In-One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer Scientific, Waltham, MA, USA).
(Other CRISPRs that worked, but were less effective, were 5'-GTATACTCACGCCACCCAC (CGG) -3 'and 5'-GGCGTATGTATCAGTCTCAG (TGG) -3'). The miPSC were transfected with AIO vectors using Neon electroporation with two pulses of 1,200 V lasting 20 ms. The transfected iPSC cultures were dissociated into single cells using 0.05% Trypsin (Gibco) and then classified with the
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80/105 FACSAria TM cells (BD Bioscience, Franklin Lakes, NJ, USA) to remove doublets and debris by selective selection in the emission of frontal and lateral dispersion. The individual cells were expanded into full-size colonies and tested for CRISPR editions by scanning for the presence of the aberrant sequence of the CRISPR dividing site. Briefly, the target sequence was amplified via PCR using AmpliTaq Gold Mastermix (Thermo Fisher-Applied Biosystems, Waltham, MA, USA) and B2m gDNA primers:
[0217] F: 5'- CTGGATCAGACATATGTGTTGGGA-3 ', [0218] R: 5'-GCAAAGCAGTTTTAAGTCCACACAG-3' [0219] After cleaning the obtained PCR product (PureLink ® Pro 96 PCR Purification Kit, Thermo Fisher Scientific , Waltham, MA, USA), Sanger sequencing was performed using an Ion Personal Genome Machine (PGM ™, Thermo Fisher Scientific). The sequencing to identify homogeneity, a 250 bp region of the B2m gene, was amplified by PCR using B2m gDNA PGM primers:
[0220] F: 5’-TTTTCAAAATGTGGGTAGACTTTGG-3 'and [0221] R: 5' - GGATTTCAATGTGAGGCGGGT-3 '.
[0222] The PCR product was purified as previously described and prepared using the Lon PGM Hi-Q Model Kit (Thermo Fisher Scientific). The experiments were carried out on the Ion PGM ™ System with the Lon 318 ™ Chip Kit (Thermo Fisher Scientific). Pluripotency analyzes were performed again.
[0223] As seen in Figure 4, β2-microglobulin expression was eliminated in miPSC cells. MHC-I expression was not induced by IFN-γ stimulation (panel
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81/105 on the right). As a control, the parental miPSC cells were stimulated with IFN-γ (left panel).
ç. EXAMPLE 2: GENERATION OF PLURIPOTENT CELLS SUBMITTED TO DOUBLE KNOCKOUT β-2 MICROGLOBULIN / CIITA [0224] CRIPSR technology was used for the additional knockout of the Ciita gene. To target the coding sequence of the mouse Ciita gene, the CRISPR 5'-GGTCCATCTGGTCATAGAGG (CGG) -3 'sequence was paired and ligated into the All-In-One (AIO) vectors containing the Cas9 expression cassette according to the instructions of the kit (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer, Waltham, MA, USA). miPSC were transfected with AIO vectors using the same condition for B2m-K0. The transfected iPSC cultures were dissociated into individual cells using 0.05% Trypsin (Thermo Fisher-Gibco) and then separated with FACSAria ™ cell separator (BD Bioscience, Franklin Lakes, NJ, USA) to remove doublets and debris by selective classification in emission by frontal and lateral dispersion. The individual cells were expanded into full-size colonies and tested for CRISPR editions by scanning for the presence of the aberrant sequence of the CRISPR dividing site. Briefly, the target sequence was amplified via POR with the use of AmpliTaq Gold Mastermix (Thermo Fisher Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA F: 5'-IncorporaçõesCAGAACGATGAGCTT3 ', R: 5’-TGCAGAAGTCCTGAGAAGGCC-3' primers. After cleaning the obtained PGR product (PureLink® Pro 96 PGR Purification Kit, Thermo Fisher, Waltham, MA, USA), Sanger sequencing was performed. Using the DNA sequence chromatogram, the edited clones were then identified through the presence of the aberrant sequence of the CRISPR dividing site. The size
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82/105 of indel was calculated using the TIDE tool. PCR and ICC were performed again to check the pluripotency status of the cells.
[0225] Figure 5 confirms the double knockout of miPSC / B-2-microglobulin / Ciita. MHC-II cannot be induced by TNF-oc to express MHC-II.
D. EXAMPLE 3: GENERATION OF CD47 + PLURIPOTENT CELLS DOUBLE β-2 MICROGLOBULIN / CIITA KNOCKOUT [0226] A Cd47 expression vector was introduced in the B2m / Ciita double elimination miPSC generated above. The vector was delivered using lentivirus containing the antibiotic resistance cassette Blasticidin. The sequence of the Cd47 gene was synthesized and the DNA was cloned in the plasmid Lentivirus pLenti6 / V5 (ThermoFisher, Waltham, MA, USA) containing a marker of resistance to blasticidin. Sanger follow-up was performed to verify that no mutations occurred. The generation of lentiviruses was performed with a 1 x 10 ⁷ TU / ml stucco titer. The recombinant vector was transduced to 2x10 ⁵ B2M-KO / CIITA double knockout mIPSCs, cultured in blasticidine MEE resistant cells for 72 h with an MOI ratio of 1:10 followed by antibiotic selection with 12.5 ug / ml blasticidine by 7 days. The banks selected with antibiotics were tested by RT-gPCR amplification of Cd47 mRNA and detection by Cd47 flow cytometry. After confirming Cd47 expression, the cells were expanded and subjected to pluripotency assays.
[0227] Figure 6A shows the increased Cd47 expression of a transgene added to the β2-microglobulin / Ciita double knockout (iPS ^hypo cells). Figure 6B shows how hypo C57BL / 6 iPS cells survive in the environment
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83/105 allogeneic BALB / c, but parental IPS cells do not. This new result confirms that hypoimmune pluripotent cells survive when transplanted into incompatible hosts.
E. MHIP CELL MOUSE CELL DIFFERENTIATION [0228] Islet cells: mHIP cells were differentiated into islet cells using techniques adapted from Liu et al. , Exp. Diabetes Res 2012: 201295 (doi: 10.1155 / 2012/201295), incorporated herein by reference and in particular for the differentiation techniques presented in it. The iPS cells were transferred to gelatin-coated flasks for 30 min to remove the feeder layer and seeded at 1 x 10 ⁶ cells per well to collagen I-coated plates in DMEM / F-12 medium supplemented with 2 mM glutamine, a non-essential amino acid. 100 μΜ, 10 ng / ml of activin A, 10 mM nicotinamide and 1 pg / ml of laminin with 10% FBS overnight. ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 μΜ non-essential amino acids, 10 ng / ml activin A, 10 mM nicotinamide, 25 pg / ml insulin and 1 pg / ml laminin with 2% FBS for 6 days.
[0229] Neural stem cells: mHIP cells were differentiated into neural cells using techniques adapted from Abraches et al., Doi: 10.1371 / journal.pone. 0006286, here incorporated by reference and in particular for the differentiation techniques presented in it. To start the monolayer protocol, ES cells were plated in ESGRO Complete Clonal Grade medium serum medium (Millipore) at high density (1.5 χ 10 ⁵ cells / cm ² ). After 24 hours, ES cells were gently dissociated and
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84/105 plated on tissue culture plastic coated with 0.1% (v / v) gelatin at 1 x 10 ⁴ cells / cm ² in RHB-A or N2B27 medium (StemCell Science Inc.), changing the medium each other. day. For replating on day 4, cells were dissociated and plated at 2 x 10 ⁴ cells / cm ² in plastic culture tissue coated with laminin in medium supplemented with RHB-A with 5 ng / ml of murine bFGF (Peprotech). From that point, the cells were replanted in the same conditions every 4 days and the medium was changed every 2 days, totaling 20 days in culture. To quantify the number of differentiating neurons at each point in time, cells were plated on laminin coated glass slides in 24 wells, Nunc plates and, 2 days after plating, the medium was changed to a mixture of RHB-A: Neurobasal: B27 (1: 1: 0.02), to allow better survival of differentiated neurons.
[0230] Smooth muscle cells: mHIP cells were differentiated into SM cells using techniques adapted from Huang et al., Blochem Blophys Res Commun 2006: 351 (2) 321-7, incorporated herein by reference and in particular for the techniques of differentiation described there. The resuspended iPSCs were grown in plastic petri dishes coated with 6-well gelatin (Falcon, Becton-Dickinson) at 2 my cells per well at 37 ° C, 5% CO2 in 2 ml of differentiation medium with the presence of atRA 10 μΜ, respectively. The differentiation medium was produced from DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin and streptomycin. The culture was continued for 10 days with a daily change of fresh media.
[0231] From the 11th day, the means of
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85/105 differentiation was replaced by serum-free culture medium, consisting of knockout DMEM, replacement of 15% knock-out serum, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin and streptomycin. Cultures were continued for another 10 days with daily change of serum-free medium.
[0232] Cardiomyocytes: mHIP cells were differentiated into CM cells using techniques adapted from Kattman et al., Cell Stem Cell 8: 228-240 (2011), incorporated by reference and in particular for the differentiation techniques presented the same.
[0233] Endothelial cells: mHIP cells were differentiated into endothelial cells, as is known.
F. EXAMPLE 4: ALLOGENIC TRANSPLANTATION OF HIP CELL DERIVATIVES SHOWS LONG-TERM SURVIVAL IN TOTALLY IMMUNOCOMPETENT RECEPTORS
The. MICE:
[0234] BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU, CAnN.CgFoxnl // Crl, H2d) and beige Scid (CBySmn. CB17-Prkdcscid / J) (all 6-12 weeks) were used as recipients for different assays (all 6-12 weeks of life). The number of animals per experimental group is shown in each Figure. The mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and received humanitarian care according to the Guide for Principles of Laboratory Animals. Animal experiments were approved by the Amt fur Gesundheit und Verbraucherschutz in Hamburg and carried out in accordance with EU and local guidelines.
B. PLURIPOTENCE ANALYSIS BY RT-PCR AND IF:
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86/105 [0235] miPSC were plated on 24-well plates and processed for RT-PCR and immunofluorescence (IF) 48 h after plating. For the ICC, the cells were fixed, permeabilized and blocked using the Image-iT ™ Fixation / Permeabilization Kit (Thermo Fisher n ° Cat., R37602). The cells were stained overnight at 4 ° C with primary antibodies to Sox2, SSEA-1, Oct4 and Alkaline Phosphatase. After several washes, the cells were incubated with a secondary AlexaFluor 488 antibody and NucBlue Fixed Cell ReadyProbes reagent (all Thermo Fisher Scientific). The spotted cells were photographed using a fluorescent microscope.
[0236] For RT-PCR, RNA was extracted using the PureLink ™ RNA Mini Kit (Thermo Fisher No. Cat. 12183018A). A DNase I step was included to remove the contaminating genomic DNA. The cDNA was generated using Applied Biosystems® High Capacity cDNA Reverse Transcription Kit. Controls without reverse transcriptase (without RT) were also generated from all RNA samples. Specific primers for the gene were used to amplify the target sequences with the use of AmpliTaq Gol ® 360 Master Mix (Thermo Fisher n ° Cat. 4398876). The PCR reactions were visualized on 2% agarose gels. A set of positive control primers has been included that amplifies a constitutively expressed home maintenance gene (Actb) that encodes a cell cytoskeleton protein.
ç. GENERAL EDITION OF MICE IPSCS:
[0237] miPSCs were submitted to 3 gene editing steps. First, CRISPRs targeting the coding sequence of the mouse B2m gene were
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87/105 paired and linked in vectors containing the Cas9 expression cassette. The transected miPSCs were dissociated in individual cells, expanded into colonies, sequenced and tested for homogeneity. Second, these B2m - / - miPSCs were transected with vectors containing CRISPRs targeted at Ciita, the main regulator of MHO II molecules. The colonies of individual expanded cells were sequenced and the B2m - / - Ciita - / - clones were identified through the presence of an aberrant sequence from the CRISPR dividing site. Third, the Cd47 gene sequence was synthesized and the DNA was cloned into a plasmid lentivirus with a resistance to blasticidin. The B2m - / - Ciita- / miPSCs were transected and cultured in the presence of blasticidin. The groupings selected with antibiotics were tested for Cd47 overexpression and the B2m - / - Ciita - / - Cd47tg miPSCs were expanded. FACS analyzes showed high expression of MHO I, modest but detectable expression of MHO II and negligible expression of Cd47 in miPSCs wt. The lack of MHO I expression, MHO II expression and Cd47 overexpression in the associated created miPSC lines was confirmed. All projected miPSC lines were tested for pluripotentiality. This was confirmed in miPSCs B2m- / Ciita - / - Cd47tg after 3 stages of manipulation and its potential to form cells from all 3 germ layers.
d. MIPSCS GENERATION B2M- / ~:
[0238] CRIPSR technology was used to knock out the B2m gene. To target the coding sequence of the mouse beta-2-microglobulin (B2m) gene, the CRISPR 5'-TTCGGCTTCCCATTCTCCGG (TGG) -3 'sequence was paired and ligated into the All-In-One (ATO) vectors containing the
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88/105 Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer, Waltham, MA, USA). MiPSC were transfected with vectors vetores using Neon electroporation with two 1200 V pulses lasting 20 ms. The transfected iPSC cultures were dissociated into single cells using 0.05% Trypsin (Gibco) and then classified with the FACSAria cell separator (BD Bioscience, Franklin Lakes, NJ) to remove doublets and debris by classification selective emission by frontal and lateral dispersion. The individual cells were expanded into full-size colonies and tested for CRISPR editing by scanning for the presence of an aberrant sequence at the CRISPR dividing site. Briefly, the target sequence was amplified by PCR using AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and the B2M ADNg F: 5'CTGGATCAGACATATGTGTTGGGA-3 ', R: 5'-GCAAAGCAGTTTAAGTACTACTACAGACACACTACTACAGACACACACTACTACTACAGACACCACTACTACTACTACAGACACC After cleaning the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. With the Ion Personal Genome Machine (PGM) to identify homogeneity, a 250 bp region of the B2m gene was amplified by PCR using the B2m gDNA PGM F primers: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3 'and R: 5'GGATTTCAATGTGAGGCGGT-3 '. The PCR product was purified as previously described and prepared using the Ton PGM Hi-Q Model Kit (Thermo Fisher). The experiments were carried out on the Ion PGM ™ System with the Ton 318 ™ Chip Kit v2 (Thermo Fisher). Pluripotency analyzes were performed again.
[0239] A reduced rate of growth or differentiation capacity of B2m iPSCs - / - was not observed
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89/105 as previously reported in the technique.
and. MIPSCS B2M GENERATION - / - AND CIITA - / -:
[0240] CRIPSR technology was used for the additional knockout of the Ciita gene. To target the coding sequence of the mouse Ciita gene, the CRISPR 5'-GGTCCATCTGGTCATAGAGG (CGG) -3 'sequence was paired and ligated into the All-In-One (AIO) vectors containing the Cas9 expression cassette according to the instructions of the kit (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer, Waltham, MA, USA). The miPSCs were transfected with the AIO vectors using the same condition for B2m-K0. The transfected miPSC cultures were dissociated into individual cells using 0.05% Trypsin (Gibco) and then separated with FACSAria cell classifier (BD Bioscience, Franklin Lakes, NJ) to remove duplicates and debris by selective emission classification by frontal and lateral dispersion. The individual cells were expanded into full-size colonies and tested for CRISPR editing by screening for the presence of aberrant CRISPR dividing site. Briefly, the target sequence was amplified via PCR with the use of AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA F: 5'-IncorporaçõesCAGAACGATGAGCTT-3 ', R: 5'TGCAGAAGTCCTGAGAAGGCC-3' primers. After cleaning the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. Using the DNA sequence chromatogram, the edited clones were then identified through the presence of an aberrant sequence from the CRISPR dividing site. The size of the indel was calculated using the TIDE tool. PCR and ICC were performed again to check the pluripotency status
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90/105 of the cells.
f. MIPSCS GENERATION B2M - / - CIITA - / - CD47TG:
[0241] The iPSC cell line B2m-KO, Ciita-KO and Cd47-tg was generated by antibiotic resistance selection after lentivirus-mediated administration of a Cd47 expression vector containing the antibiotic resistance cassette Blasticidin. The Cd47 gene sequence was synthesized and the DNA was cloned into the plasmid Lentivirus pLenti6 / V5 (ThermoFisher) with a resistance to blasticidin. Sanger sequencing was performed to verify that no mutations occurred. The generation of lentiviruses was performed with a stock titre of 1 x 10 ⁷ TU / ml. Transduction was performed in 2x10 ⁵ miPSCs B2m - / - Ciitas - / -, cultured in MEE cells resistant to blasticidin for 72 h with a 1:10 MOI ratio followed by antibiotic selection with 12.5 pg / ml of Blasticidin by 7 days. Banks selected with antibiotics were tested by RT-qPCR amplification of Cd47 mRNA and detection by Cd47 flow cytometry. After confirming Cd47, the cells were expanded and confirmed by pluripotency tests.
g. DERIVATION AND CHARACTERIZATION OF iIPSC-DERIVED ENDOTHELIAL CELLS (iECs):
[0242] The iECs were derived using a three-dimensional approach. Briefly, to initiate differentiation, iPSCs were cultured on ultra-low non-adhesive plates to form embryonic body (EB) aggregates in EBM2 medium (Lonza) in the absence of leukemia inhibiting factor (LEL). After 4 days of suspension culture, the EBs were fixed again in plates coated with 0.2% gelatin and cultured in EBM2 medium supplemented with VEGF-A165 (50 ng / ml;
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PeproTech). After 3 weeks of differentiation, individual cell suspensions were obtained using a cell dissociation buffer (Life Technologies) and stained with APC-conjugated anti-mouse antibodies (eBiosciences) and PE-conjugated CD144 (BD Biosciences). The iECs were purified by fluorescence-activated cell screening (FACS) of the CD31 + CD144 + population. IECs were maintained in EBM2 medium supplemented with recombinant murine vascular endothelial growth factor (50 ng / ml).
[0243] Its phenotype was confirmed by immunofluorescence for CD31 and VE cadherin, as well as by PCR and tube formation assays to demonstrate endothelial function to form premature vascular structures. Note: the differentiation protocols with the use of 1PSC confluent monolayers in 0.1% gelatin or Matrigel were also successful. Note: other endothelial cell media have also been used successfully.
H. DERIVATION AND CHARACTERIZATION OF SMOOTH MUSCLE CELLS DERIVED FROM 1PSC (iSMCs):
[0244] Resuspended IPSCs were cultured in 6-well Petri dishes, coated with 0.1% gelatin (Falcon, Becton-Dickinson) at 2 medium cells per well at 37 ° C, 5% CO2 in 2 ml differentiation medium with the presence of 10 µM. The differentiation medium was performed using DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin and streptomycin. The culture was continued for 10 days with daily changes of the medium.
[0245] From the 11th day, the differentiation medium was replaced by a culture medium free of
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92/105 serum from a knockout DMEM: replacement of 15% knockout serum, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin and streptomycin. Cultures were continued for another 10 days with daily changes to the serum-free medium. The phenotype was confirmed by immunofluorescence and PCR for both SMA and SM22.
i. DERIVATION AND CHARACTERIZATION OF iPSC DERIVED CARDIOMYOCYTES (iCMs):
[0246] Before differentiation, iPSCs were passed twice on gelatin-coated plates to remove feeder cells. Briefly, iPSCs were dissociated with TrypLE (Invitrogen) and cultured at 75,000 to 100,000 cells / ml without any additional growth factors for 48 hours. The 3-day-old EBs were dissociated and the cells were differentiated under cardiac conditions. In summary, 6 χ 10 ⁴ to 10 χ 10 ⁴ cells were seeded in individual wells of a 96-well flat-bottom plate (Becton Dickenson, Franklin Lakes, NJ, USA) coated with gelatin in StemPro-34 SF (Invitrogen) , supplemented with 2 mM L-glutamine, 1 mM ascorbic acid (Sigma), human VEGF (5 ng / ml), human DKK1 (150 ng / ml), human bFGF (10 ng / ml) and human FGF10 (12.5 ng / ml) (R & D Systems). The cultures were collected 4 or 5 days later (total of 7 or 8 days).
[0247] Its phenotype was confirmed by IF for troponin I and sarcomeric alpha-actinin, as well as PCR for Gata4 and Mhy6. The cells started to beat between 8-10 days. This demonstrated its functional differentiation.
j. DERIVATION AND CHARACTERIZATION OF iPSC-DERIVED ISLET CELLS (ilCs) [0248] iPS cells were transferred to
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93/105 jars coated with gelatin for 30 min to remove the feeder layer and seeded at 1 x 10 ⁶ cells per well for plates coated with collagen-I in DMEM / F-12 medium supplemented with 2 mM glutamine, 100 μΜ non-essential amino acids , 10 ng / ml activin A, 10 mM nicotinamide and 1 pg / ml laminin with 10% FBS overnight. The ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 μ não non-essential amino acids, 10 ng / ml of activin A, 0 mM nicotinamidal, 25 pg / ml of insulin and 1 pg / ml laminin with 2% FBS for 6 days. Its phenotype was confirmed by immunofluorescence for C-peptide, PCR for glucagon, Ngn3, amylase, insulin 2, somatostatin and insulin production.
k. DERIVATION AND CHARACTERIZATION OF IPSC-DERIVED NEURONAL CELLS (iNCs) [0249] To start the monolayer protocol, iPSCs were gently dissociated and plated on 0.1% of culture plastic tissue coated with gelatin at 1 x 10 ⁴ cells / cm ² in RHB-A or N2B27 media (StemCell Science Inc.), to change the media every two days. For replating on day 4, cells were dissociated and plated at 2 x 10 ⁴ cells / cm ² in plastic culture tissue coated with laminin in RHB-A medium supplemented with 5 ng / ml murine bFGF (Peprotech). From that point, the cells were replanted in the same conditions every 4 days and the medium was changed every 2 days, totaling 20 days in culture. To quantify the number of differentiating neurons at each point in time, cells were plated on laminin coated glass slides in 24 wells, Nunc plates and, 2 days after plating, the medium was changed to a mixture of
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RHB-A: Neurobasal: B27 (1: 1: 0.02), to allow better survival of differentiated neurons. Its phenotype was confirmed by IF for Tuj-1 and nestin.
l. ELISPOT ASSAYS [0250] For the EnmuneLinked ImmunoSpot (Elispot) unidirectional assays, recipient splenocytes were isolated from the fresh spleen 5 days after cell injection (miPSC, miPSC B2m - / - or miPSC B2m - / - or miPSC B2m - / - Ciita / - Cd47tg) and used as responder cells. Donor cells (miPSC, miPSC B2m - / - or miPSC B2m - / - Ciita - / - or miPSC B2m- / Ciita - / - Cd47tg) were inhibited by mitomycin and served as stimulator cells. 10 ⁶ stimulator cells were incubated with 5x10 ⁵ receptor splenocytes for 24 h and IFNy and IL-4 spot frequencies were automatically enumerated using an ELISPOT plate reader. Quadruplicates were performed in all trials.
m. TERATOMA TESTS TO STUDY Ipsc SURVIVAL IN VIVO [0251] Six week old singenic or allogeneic mice were used for transplantation of non-immunogenic wtiPSCs or iPSCs. Ix10 ⁶ cells were injected in 100 ul into the right thigh muscle of mice. The transplanted animals were observed routinely every two days, and the growth of the tumor was measured with a caliper. They were sacrificed after the development of tumors larger than 1.5 cm ³ or after an observation period of 100 days.
n. NK IN VITRO CELL TESTS [0252] CD107 expression in NK cells after co-culture with wtiPSCs or HIP cells was measured by
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95/105 flow cytometry as a marker of NK cell activation. Using the Elispot principle, NK cells were co-cultured with wtiPSCs or HIP cells and their de-γ release was measured.
[0253] According to the missing self theory, MHC I-deficient stem cells have been shown to be susceptible to death by NK, since both murine and human PSCs express ligands to activate NK receptors. Although it has been reported that the expression of activating receptors decreases with differentiation, the death of NK from B2m - / - derivatives has been observed. Although isolated expression of HLAE or HLA-G in human pluripotent stem cells has been used to mitigate the expected innate immune response in HLA I - / - cells, there are additional very effective non-MHC inhibitor ligands between them. The invention reports that Cd47 was considered a surprisingly potent inhibitor of innate immune clearance.
O. SUMMARIES OF MOUSE DATA [0254] All projected miPSC lines were transplanted into C57BL / 6 singenic and allogeneic BALB / c receptors without any immunosuppression. Although all manipulated cells similarly developed teratomas at syngeneic receptors, their survival depended on their level of hypoimmunogenicity at allogeneic receptors. The formation of 60% of B2BC - / - miBC teratomas in BALB / c, a subtle Elispot response and a measurable IgM antibody response was observed. In B2PS - / - Ciita - / - miPSCs, 91.7% teratoma formation was observed in allogeneic BALB / c, a minor Elispot response, and no antibody response. The miPSC B2m - / - Ciita - / - Cd47tg final line showed 100%
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96/105 teratoma and no Elispot or antibody response. The contribution of Cd47 overexpression was further evaluated in innate immunity assays, comparing B2m - / - Ciita - / - miPSCs with B2m - / - Ciita - / - Cd47tg miPSCs. Overexpression of CD47 significantly reduced the expression of CD107 from NK cells and the release of ΙΕΝ-γ from NK cells, thus mitigating innate immune clearance. In summary, each stage of manipulation has made miPSCs more hypoimmunogenic.
[0255] B2m - / - Ciita - / - HIP cells differentiated into hypoimmunogenic endothelial-like cells (miECs), smooth muscle-like cells (miSMCs) and cardiomyocyte-like cells (miCMs). Derivatives of wild-type miPSC (that is, unprocessed miPSCs) served as controls. All derivatives showed the typical morphological aspect, the immunofluorescence of the cell marker and the genetic expression of their intended mature tissue cell lines. The expression of MHC I and II molecules in wt derivatives was generally largely upregulated compared to their parental miPSC line, but markedly varied by cell type. As expected, miECs had by far the highest expression of MHC I and MHC II, miSMCs had moderate expression of MHC I and MHC II, while miCMs had moderate expression of MHC I, but very low MHC II. All wt derivatives had very low Cd47 expression, although also slightly higher than that of miPSCs. All B2m - / - Ciita - / - Cd47tg derivatives appropriately showed a complete lack of MHC I and MHC II and Cd47 significantly greater than their wt counterparts.
[0256] Matrigel plugs containing 5x10 ⁵ mi miECs
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97/105 wt, miSMCs and miCMs were transplanted into subcutaneous pouches of singenic C57BL / 6 or allogeneic BALB / c mice. After 5 days, all allogeneic receptors mounted a strong cellular immune response, as well as a strong IgM antibody response against these wild-type cell grafts. In sharp contrast, none of the corresponding B2m- / Ciita - / - Cd47tg (HIP) derivatives showed detectable increases in ΙΕΝ-γ Elispot customers or IgM antibody production.
[0257] The morphology of the transplanted cells has also been confirmed. Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 myelo cells, MIPSC-derived cardiomyocytes (miCM), MIPSC-derived smooth muscle cells (miSMC) or miPSC-derived endothelial cells (miEC) in 250 ul 0.9% saline were mixed with 250 ul BD Matrigel High Concentration (1: 1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10 and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin inclusion. . 5 pm thick sections were cut and stained with hematoxylin and eosin (HE). Histology confirmed miCMs, miSMCs and morphologically adherent miECs.
g. EXAMPLE 5: GENERATION OF HUMAN IPSCS [0258] The Human iPSC Epissomal Line was derived from CD34 + cord blood (Cat. No. A33124, Thermo Fisher Scientific) using an EBNA-based episomal system of three plasmids, seven factors (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4,
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MYC, NANOG, LIN28 and Antigen SV40L T) from ThermoFisher. This iPSC line is considered to have a zero footprint due to the fact that there was no integration into the genome from the reprogramming event. It has been shown to be free of all reprogramming genes. IPSCs have a normal XX karyotype and an endogenous expression of pluripotent markers such as Oct4, Sox2, Nanog (as shown by RT-PCR) Oct4, SSEA4, TRA-1-60 and TRA-1-81 (as shown by ICC). In targeted differentiation and teratoma analyzes, these hiPSCs maintained their differentiation potential for ectodermal, endodermal and mesodermal strains. In addition, vascular, endothelial and cardiac lines were derived with robust efficiencies.
[0259] Note: several gene delivery vehicles for generating iPSC have been used successfully, including retroviral vectors, adenoviral vectors, Sendai virus, as well as virus-free reprogramming methods (using episomal vectors, piggyBac transposon, synthetic mRNAs, microRNAs, recombinant proteins and small molecule drugs, etc.).
[0260] Note: different factors have been used successfully for reprogramming, such as the first reported combination of OCT3 / 4, SOX2, KLF4 and C-MYC, known as Yamanaka factors. In one modality, only three of these factors were successfully combined and omitted C-MYC, although with reduced reprogramming efficiency.
[0261] In one embodiment, L-MYC or GLIS1 instead of C-MYC showed better reprogramming efficiency. In another modality, reprogramming factors are not limited to genes associated with pluripotency.
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The. STATISTICS [0262] All data are expressed as mean ± SD or in box plots with the median and minimum range to maximum. Intergroup differences were adequately assessed by unpaired Student's t test or by one-way analysis of variance (ANOVA) with Bonferroni's postHoc test. * p <0.05, ** p <0.01.
H. EXAMPLE 6: HIP HUMAN CELL GENERATION [0263] IPSC Cas9 Human was subjected to 2 stages of gene editing. In the first stage, CRISPR technology was performed by a combined targeting of the coding sequence of the human beta-2-microglobulin (B2M) gene with the CRISPR 5'-CGTGAGTAAACCTGAATCTT-3 'sequence and the coding sequence of the human CIITA gene with the sequence CRISPR 5'GATATTGGCATAAGCCTCCC-3 'linearized with the T7 promoter was used to synthesize the gRNA according to the kit instructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). The collected in vitro transcription gRNA (IVT) was then purified using the MEGAclear Transcription Cleaning Kit. For IVT RNA administration, the singled cells were electroporated with 300 ng IVT gRNA using a Neon electroporation system. After electroporation, the edited Cas9 iPSCs were expanded to inoculate single cells: iPSC cultures were dissociated in isolated cells using TrypLE (Gibco) and stained with Alexa Fluor 488's Tral-60 and propidium iodide (PI). The cell classifier FACS Aria (BD Biosciences) was used for the classification and the doublets and debris were excluded from sowing by selective classification in the emissions by frontal and lateral dispersion. Viable pluripotent cells
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100/105 were selected in the absence of PI and presence of staining of Tral-60 Alexa Fluor 488. The individual cells were then expanded into full-size colonies, after which the colonies were tested for a CRISPR edition. CRISPR-mediated divage was assessed using the GeneArt Genomic Cleavage Detection Kit (Thermo Fisher). Genomic DNA was isolated from Ix10 ⁶ hiPSCs and CIITA genomic DNA regions from B2M and was amplified by PCR using AmpliTaq Gold 360 Master Mix and F: 5'TGGGGCCAAATCATGTAGACTC-3 'and R: 5'- primer sets TCAGTGGGGGTGAATTCAGTGT-3 'for B2M as well as F: 5' -CTTAACAGCGATGCTGACCCC-3 'and R: 5'TGGCCTCCATCTCCCCTCTCTT-3' for CIITA. For TIDE analysis, the PCR product obtained was cleaned (PureLink PCR Purification Kit, Thermo Fisher) and Sanger sequencing was performed to predict indel frequency. After confirming the B2M / CIITA knockout, the cells were further characterized by analyzing the karyotype and the TaqMan hPSC Classification Panel (Thermo Fisher). PSC was considered pluripotent and maintained a normal karyotype (46, XX) during the genome editing process.
[0264] In the second step, the CD47 gene was synthesized and the DNA was cloned into a plasmid lentivirus with an EFla promoter and puromycin resistance. The cells were transduced with lentiviral stocks of Ix10 ⁷ TU / ml and 6 qg / ml of Polybrene (Thermo Fisher). The medium was changed daily after transduction. Three days after transduction, the cells were expanded and selected with 0.5 qg / ml of puromycin. After 5 days of antibiotic selection, antibiotic resistant colonies appeared and were expanded to generate stable clusters. The CD47 level was confirmed by qPCR.
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Pluripotentiality assay (TaqMan hPSC Classification Panel, Thermo Fisher) and karyotyping were performed again to check the pluripotent status of the cells.
I. EXAMPLE 7: HIP HUMAN CELL DIFFERENTIATION
1. HHIP CELL DIFFERENTIATION FROM HUMAN CARDIOMYOCYTES [0265] This was accomplished using a protocol adapted from Sharma et al., J. Vis Exp. 2015 doi: 10.3791 / 52628, incorporated herein by reference in its entirety and specifically for techniques for differentiate cells. HiPSCs were plated in diluted Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8 Flex medium (Thermo Fisher). After the cells reached 90% confluence, differentiation was initiated and the medium was changed to 5 ml of RPMI1640 containing 2% B-27 minus Insulin (both Gibco) and 6 µM CHIR-99021 (Selleck Chem). After 2 days, the medium was changed to RPMI1640 containing 2% B-27 minus insulin without CHIR. On day 3, 5 µl of IWR1 was added to the medium for two more days. On day 5, the medium was switched back to RPMI 1640 containing B-27 medium at 2% less insulin and incubated for 48 h. On day 7, the medium was changed to RPMI 1640 containing B27 plus insulin (Gibco) and replaced every 3 days afterwards with the same medium. The spontaneous beating of cardiomyocytes was visible for the first time at approximately day 10 to day 12. Purification of cardiomyocytes was performed on day 10 after differentiation. Briefly, the medium was switched to low glucose medium and maintained for 3 days. On day 13, the medium was switched back to RPMI 1640 containing B27 plus insulin. This procedure was repeated on day 14. The remaining cells are highly purified cardiomyocytes.
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2. HHIP CELL DIFFERENTIATION FROM HUMAN ENDOTHELIAL CELLS [0266] HiPSC were plated in diluted Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8 Flex medium (Thermo Fisher). After the cells reached 60% confluence, differentiation was initiated and the medium was changed to RPMI1640 containing 2% B-27 minus insulin (both from Gibco) and CHIR-99021 5 μΜ (Selleck Chem). On day 2, the medium was switched to reduced medium: RPMI1640 containing 2% B-27 minus insulin (both from Gibco) and CHIR-99021 2 μΜ (Selleck Chem). From day 4 to day 7, cells were exposed to RPMI EC, RPMI1640 medium containing 2% B-27 minus Insulin plus 50 ng / ml vascular endothelial growth factor (VEGF; R & D Systems, Minneapolis, MN, USA ), 10 ng / ml basic fibroblast growth factor (FGFb; R & D Systems), Y27632 10 μΜ (Sigma-Aldrich, Saint Louis, MO, USA) and SB 431542 1 μΜ (Sigma-Aldrich). Clusters of endothelial cells were visible from day 7 and cells were maintained in EGM-2 SingleQuots (Lonza, Basel, Switzerland) plus 10% FCS hi (Gibco), 25 ng / ml of endothelial vascular growth factor (VEGF; R & D Systems, Minneapolis, MN, USA), 2 ng / ml basic fibroblast growth factor (FGFb; R & D Systems), Y-27632 10 μΜ (Sigma-Aldrich, Saint Louis, MO, USA) and SB 431542 1 μΜ (Sigma-Aldrich). The differentiation process was completed after 14 days and undifferentiated cells highlighted during the differentiation process. For purification, the cells went through the progress of MACS according to the manufacturer's protocol using CD31 microspheres (Miltenyi, Auburn, CA). Highly purified EC cells were cultured in EGM-2 SingleQuots (Lonza, Basel, Switzerland)
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103/105 plus supplements and FCS hi 10% (Gibco). TrypLE was used to pass cells 1: 3 every 3 to 4 days.
j. TRANSPLANTATION IN HUMANIZED MICE [0267] Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). 1 myo cells, or hiPSC-derived cardiomyocytes (hiCM) or hiPSC-derived endothelial cells (hiECin 250 ul 0.9% saline solution containing ZVAD (100 mM, benzyloxycarbonyl-Val-Ala-Asp (O-methyl) -fluoromethylketone , Calbiochem), Bcl-XL BH4 (TAT peptide permeated by cells, 50 nM, Calbiochem), cyclosporin A (200 nM, Sigma), IGF-1 (100 ng / ml, Peprotech) and pinacidil (50 mM, Sigma) were mixed with 250 µl BD Matrigel High Concentration (1: 1; BD Biosciences) and injected subcutaneously into the lower dorsal of mice using a 23-G syringe. Matrigel plugs were explanted 2, 4, 6, 8, 10 and 12 weeks after the implant and were fixed with paraformaldehyde 4% and glutenaldehyde 1% for 24 hours, followed by dehydration and inclusion in paraffin, a section of 5 pm thick was cut and stained with Hematoxylin and Eosin (HE) and the morphology was confirmed.
ix. EXAMPLE SEQUENCES:
SEQ ID NO: - 1 Human β-2-Microglobulin [0268] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSN FLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYR
SEQ ID NO: 2 - Human CIITA protein, 160 amino acid N-terminal [0269] MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSD ADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELD
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QYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKP
SEQ ID NO: 3 - Human CD47 [0270] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTWIPC FVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLH YYVFS Tai GLT S EVI I AY LVIQVI ILAWGLS LCIAACIPMHGPLLIS GLSILALAQLLG LVYMKFVE
SEQ ID NO: 4 - Herpes Virus Thymidine Kinase
Simplex (HSV-tk) [0271] MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVR
LEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYT TQHRLDQGEISAGDAAWMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDR HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPG ERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIG DTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSG MVQTHVT T PGSIPTICDLART FAREMGEAN
SEQ ID NO: 5 - Escherichia coli cytosine deaminase (EC-CD) [0272] MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMP
ITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVK QRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSY PNGEALLEEALRLGADWGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQS RFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQG RFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQ INDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIAS TQPAQTTVYLEQPEAIDYKR
SEQ ID NO: 6 - Truncated human caspase 9 [0273] GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNECRES
GLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILS
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HGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFE VASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPK SGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS [0274] All publications and patents disclosed or said documents are incorporated herein by reference in its entirety. The previous description has been presented for illustration and description purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims.

权利要求:
Claims (58)
[1]
1. METHOD FOR GENERATING A PLURIPOTENT STEM CELL
HYPOIMUNOGENIC, characterized by understanding:
The. eliminate the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC);
B. eliminating the activity of both alleles of a CIITA gene in said iPSC; and
ç. increase the expression of CD47 in said iPSC.
[2]
2. METHOD according to claim 1, characterized in that said iPSC is human, said B2M gene is human, said CIITA gene is human and said increase in CD47 expression results from the introduction of at least one copy of a gene Human CD47 under the control of a promoter in said cell iPSC.
[3]
3. METHOD according to claim 1, characterized in that said iPSC is murine, said B2m gene is murine, said Ciita gene is murine and said increase in Cd47 expression results from the introduction of at least one copy of a gene Murine cd47 under the control of a promoter in said iPSC cell.
[4]
METHOD according to either of claims 2 or 3, characterized in that said promoter is a constitutive promoter.
[5]
5. METHOD, according to any one of claims 1 to 3, characterized by the said interruption in both alleles of said B2M gene resulting from a reaction of Repeated Regularly Spaced Clustered Palindromes) / Cas9 (CRISPR) that breaks both alleles of the said B2M gene.
[6]
6. METHOD, according to any of the
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2/13 claims 1 to 3, characterized in that said interruption in both alleles of said CIITA gene results from a CRISPR reaction that disrupts both alleles of the CIITA gene.
[7]
7. HUMAN HYPOIMMUNOGENIC PLURIPOTENT STEM CELL (hHIP), characterized by comprising:
The. one or more changes that inactivate the two alleles of an endogenous B2M gene;
B. one or more changes that inactivate both alleles of an endogenous CIITA gene; and
ç. a change causing an increased expression of a CD47 gene in said hHIP stem cell;
wherein said hHIP stem cell triggers a first Natural Exterminating Cell (NK) response that is less than a second NK cell response induced by an induced pluripotent stem cell (iPSC) comprising said B2M and CIITA changes, but it does not comprise said increased expression of the CD47 gene and wherein said first and second NK cell responses are measured by determining the ΙΕΝ-γ levels of NK cells incubated with any of said hHIP or iPSC in vitro.
[8]
8. HUMAN HYPOIMMUNOGENIC PLURIPOTENT STEM CELL (hHIP), characterized by comprising:
The. one or more changes that inactivate the two alleles of an endogenous B2M gene;
B. one or more changes that inactivate both alleles of an endogenous CIITA gene; and
ç. one or more changes causing an increased expression of a CD47 gene in said hHIP stem cell;
in which the said hHIP stem cell triggers a first T-cell response in a mouse strain
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3/13 humanized which is less than a second T cell response in said humanized mouse strain triggered by an iPSC and wherein said first and second T cell responses are measured by determining the IFN-γ levels of said humanized mice in an Elispot trial.
[9]
9. METHOD, characterized by comprising the hHIP stem cell transplantation as defined in any of claims 7 or 8 in a human individual.
[10]
10. HYPOIMMUNOGENIC PLURIPOTENT CELL, characterized by comprising:
The. an endogenous Class I Histocompatibility Antigen (HLA-I) function that is reduced when compared to a pluripotent progenitor cell;
B. an endogenous function of Major Histocompatibility Class II Antigen (HLA-II) which is reduced when compared to said progenitor pluripotent cell; and
ç. a reduced susceptibility to death of NK cells when compared to said pluripotent parent cell;
wherein said hypoimmunogenic pluripotent cell is less susceptible to rejection when transplanted to an individual as a result of said reduced HLA-I function, wherein said HLA-II function is reduced and susceptibility is reduced to the death of NK cells.
[11]
11. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 10, characterized in that said HLAI function is reduced by a reduction in the expression of the β-2 microglobulin protein.
[12]
12. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 11, characterized in that a gene encoding said β-2 microglobulin protein is subjected to knockout.
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[13]
13. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 12, characterized in that said β-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1.
[14]
14. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 13, characterized in that said β-2 microglobulin protein has the sequence of SEQ ID NO: 1.
[15]
15. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 10, characterized in that said HLAI function is reduced by a reduction in the expression of the HLA-
THE.
[16]
16. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 15, characterized by a gene encoding said HLA-A protein to be knocked out.
[17]
17. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 10, characterized in that said HLAI function is reduced by a reduction in the expression of the HLA-
B.
[18]
18. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 17, characterized in that an HLA-B protein is knocked out.
[19]
19. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 10, characterized in that said HLAI function is reduced by a reduction in the expression of the HLA-
Ç.
[20]
20. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 19, characterized by a gene encoding said HLA-C protein being subjected to knockout.
[21]
21. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 20, wherein said
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5/13 hypoimmunogenic pluripotent cell is characterized by not understanding an HLA-I function.
[22]
22. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 21, characterized in that said HLA-II function is reduced by a reduction in the expression of the CIITA protein.
[23]
23. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 22, characterized in that a gene encoding said CIITA protein is subjected to knockout.
[24]
24. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 23, characterized in that said CIITA protein has at least 90% sequence identity with SEQ ID NO: 2.
[25]
25. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 24, characterized in that said CIITA protein has the sequence of SEQ ID NO: 2.
[26]
26. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 21, characterized in that said HLA-II function is reduced by a reduction in the expression of the HLA-DP protein.
[27]
27. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 26, characterized in that a gene encoding said HLA-DP protein is subjected to knockout.
[28]
28. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 21, characterized in that said HLA-II function is reduced by a reduction in the expression of the HLA-DR protein.
[29]
29. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 28, characterized in that a gene encoding said HLA-DR protein is subjected to knockout.
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[30]
30. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 21, characterized in that said HLA-II function is reduced by a reduction in the expression of the HLA-DQ protein.
[31]
31. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 30, characterized in that a gene encoding said HLA-DQ protein is subjected to knockout.
[32]
32. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 31, wherein said hypoimmunogenic pluripotent cell is characterized by not comprising an HLA-II function.
[33]
33. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 10 to 32, characterized in that said reduced susceptibility to death of NK cells is caused by an increased expression of a CD47 protein.
[34]
34. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 33, characterized by the increased expression of the CD47 protein resulting from a modification to an endogenous CD47 gene locus.
[35]
35. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 33, characterized by the increased expression of the CD47 protein resulting from a CD47 transgene.
[36]
36. HYPOIMMUNOGENIC PLURIPOTENT CELL according to any one of claims 33 to 35, characterized in that said CD47 protein has at least 90% sequence identity with SEQ ID NO: 3.
[37]
37. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 27, characterized in that said CD47 protein has the sequence of SEQ ID NO: 3.
[38]
38. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to
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7/13 with any one of claims 10 to 37, characterized in that it further comprises a suicide gene that is activated by a trigger that causes said hypoimmunogenic pluripotent cell to die.
[39]
39. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 38, characterized in that said suicide gene is a herpes simplex virus (HSVtk) thymidine kinase gene and said trigger is ganciclovir.
[40]
40. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 39, characterized in that said HSV-tk gene encodes a protein that comprises at least 90% sequence identity with SEQ ID NO: 4.
[41]
41. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 40, characterized in that said HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO: 4.
[42]
42. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 38, characterized in that said suicide gene is an Escherichia coli cytosine deaminase (EC-CD) gene and said trigger is 5-fluorocytosine (5-FC).
[43]
43. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 42, characterized in that said EC-CD gene encodes a protein that comprises at least 90% sequence identity with SEQ ID NO: 5.
[44]
44. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 43, characterized by said EC-CD gene encoding a protein comprising the sequence of SEQ ID NO: 5.
[45]
45. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to claim 38, characterized by said suicide gene
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8/13 encode an inducible Caspase protein and said trigger is a chemical dimerization inducer (ICD).
[46]
46. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 45, characterized in that said gene encodes an inducible Caspase protein that comprises at least 90% sequence identity with SEQ ID NO: 6.
[47]
47. HYPOIMMUNOGENIC PLURIPOTENT CELL according to claim 46, characterized in that said gene encodes an induced Caspase protein comprising the sequence of SEQ ID NO: 6.
[48]
48. HYPOIMMUNOGENIC PLURIPOTENT CELL, according to any one of claims 45 to 47, characterized in that said CID is AP1903.
[49]
49. METHOD TO PRODUCE A HYPOIMMUNOGENIC PLURIPOTENT CELL, characterized by comprising:
The. reduce an endogenous function of Class I Histocompatibility Antigen (HLA-I) in a pluripotent cell;
B. reduce an endogenous function of the Class II Histocompatibility Antigen (HLA-II) in a pluripotent cell; and
ç. increase the expression of a protein that reduces the susceptibility of said pluripotent cell to the death of NK cells.
[50]
50. METHOD according to claim 49, characterized in that said HLA-I function is reduced by reducing the expression of a β-2 microglobulin protein.
[51]
51. METHOD, according to claim 50, characterized in that said expression of the β-2 microglobulin protein is reduced by knockout of a gene encoding said
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9/13 β-2 microglobulin protein.
[52]
52. β-2 MICROGLOBULIN 50, characterized by said β-2 microglobulin protein having at least 90% sequence identity with SEQ ID NO: 1.
[53]
53. β-2 MICROGLOBULIN 51, characterized by said β-2 microglobulin protein having the sequence of SEQ ID NO: 1.
[54]
54. METHOD according to claim 49, characterized in that said HLA-I function is reduced by reducing the expression of the HLA-A protein.
[55]
METHOD, according to claim 54, characterized in that said expression of the HLA-A protein is reduced by knockout of a gene encoding said HLA-A protein.
56. METHOD, in a deal with the claim 49, featured for said HLA-I function be reduced by middle reduction of expression gives HLA- protein B. 57. METHOD, in a deal with the claim 56, featured for said expression of HLA-B protein to be reduced by knockout of a gene encoding said protein HLA-B. 58. METHOD, in a deal with the claim 49, featured for said HLA-I function be reduced by middle reduction of expression gives HLA- protein ç. 59. METHOD, in a deal with the claim 58, featured for said expression of HLA-C protein to be
reduced by knockout of a gene encoding said HLA-C protein.
[56]
60. METHOD, according to any of claims 49 to 59, characterized in that said hypoimmunogenic pluripotent cell does not comprise a function of
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10/13
HLA-1.
[57]
61. METHOD according to any one of claims 49 to 60, characterized in that said HLAII function is reduced by reducing the expression of a CIITA protein.
[58]
62. METHOD, according to claim 60, characterized in that said expression of the CIITA protein is reduced by knockout of a gene encoding said CIITA protein.
63. METHOD, according The claim 61, featured for said CIITA protein have at least 90 % in identity of sequence with SEQ ID NO: 2 • 64. METHOD, according The claim 63, featured for said CIITA protein have the sequence of SEQ ID NO: 2. 65. METHOD, accordingAny of them of claims 49 to 60, characterized by said function of HLA- II be reduced by reducing expression of a protein HLA- DP. 66. METHOD, according The claim 65, featured by dit the expression of HLA-DP protein to be
reduced by knockout of a gene encoding said protein
HLA-DP. 67. METHOD, according to any one of claims 49 to 60, characterized by said function of HLA- II be reduced by reducing the expression of a protein HLA- DR. 68. METHOD, according to claim 67, featured by the said expression of the HLA-DR protein to be
reduced by knockout of a gene encoding said protein
HLA-DR.
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69. METHOD according to any of claims 49-60, characterized in that said HLAII function is reduced by reducing the expression of an HLADQ protein.
70. METHOD, according to claim 69, characterized in that said expression of the HLA-DQ protein is reduced by knockout of a gene encoding said HLA-DQ protein.
71. METHOD according to any one of claims 49 to 70, characterized in that said hypoimmunogenic pluripotent cell does not comprise an HLAII function.
72. METHOD according to any one of claims 49 to 71, characterized by said increased expression of a protein that reduces the susceptibility of said pluripotent cell to phagocytosis of macrophages resulting from a modification to an endogenous gene locus.
73. METHOD according to claim 72, characterized in that said endogenous gene locus encodes a CD47 protein.
74. METHOD according to any one of claims 49 to 71, characterized by the increased expression of the protein resulting from the expression of a transgene.
75. METHOD according to claim 74, characterized in that said transgene encodes a CD47 protein.
76. METHOD according to either of claims 73 or 74, characterized in that said CD47 protein has at least 90% sequence identity with SEQ ID NO: 3.
77. METHOD, according to claim 76,
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12/13 characterized by said CD47 protein having the sequence of SEQ ID NO: 3.
78. METHOD according to any one of claims 49 to 77, characterized in that it further comprises the expression of a suicidal gene that is activated by a trigger that causes said hypoimmunogenic pluripotent cell to die.
79. METHOD according to claim 78, characterized in that said suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene and said trigger is ganciclovir.
80. METHOD according to claim 79, characterized in that said HSV-tk gene encodes a protein that comprises at least 90% sequence identity with SEQ ID NO: 4.
81. METHOD according to claim 80, characterized in that said HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO: 4.
82. METHOD according to claim 78, characterized in that said suicide gene is an Escherichia coli cytosine deaminase (EC-CD) gene and said trigger is 5 fluorocytosine (5-FC).
83. METHOD according to claim 82, characterized in that said EC-CD gene encodes a protein that comprises at least 90% sequence identity with SEQ ID NO: 5.
84. METHOD according to claim 83, characterized in that said EC-CD gene encodes a protein comprising the sequence of SEQ ID NO: 5.
85. METHOD, according to claim 78,
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13/13 characterized by said suicide gene encoding an inducible Caspase protein and said trigger being a specific chemical dimerization inducer (CID).
86. METHOD according to claim 85, characterized in that said gene encodes an inducible Caspase protein that comprises at least 90% sequence identity with SEQ ID NO: 6.
87. METHOD according to claim 86, characterized in that said gene encodes an inducible caspase protein which comprises the sequence of SEQ ID NO: 6.
88. METHOD according to any one of claims 85 to 87, characterized in that said CID is AP1903.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762445969P| true| 2017-01-13|2017-01-13|

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PCT/US2018/013688|WO2018132783A1|2017-01-13|2018-01-14|Immunoengineered pluripotent cells|

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