JP2022526218A - Generic donor stem cells and related methods - Google Patents

Abstract

Disclosed herein are generic donor stem cells and methods relating to their use and generation. The generic donor stem cells disclosed herein are useful in overcoming immune rejection in cell-based transplantation therapy. In certain embodiments, the generic donor stem cells disclosed herein are regulated for the expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant factors. There is. [Selection diagram] FIG. 1A

Description

Mutual reference to related applications This application is a continuation of US application 16 / 596,697, filing date October 8, 2019, and US application 16 / 596,697 is US application 16 /. It is a continuation application of 277,913, filing date February 15, 2019, and US application 16 / 277,913 is US provisional application No. 62 / 631,393, filing date February 15, 2018. Claim the interests of. The entire teachings of the above application are incorporated herein by reference.

Treatments that utilize cells derived from human pluripotent stem cells for transplantation have the potential to revolutionize the way the disease is treated. A major obstacle to their clinical conversion is allogeneic cell rejection by the recipient's immune system. Strategies aimed at overcoming this immune barrier include cell bank construction of cells with established HLA haplotypes (Nakajima et al., 2007; Taylor et al., 2005) and patient-specific induced pluripotency. Generation of sex stem cells (iPSC) (Takahashi et al., 2007; Yu et al., 2007) can be mentioned. However, due to multiple constraints (de Rham and Village, 2014; Tapia and Scholar, 2016), such an approach cannot be widely applied and can be easily administered to any patient in need of a cellular product. The need for "instant delivery" cell preparations only stands out.

Disclosed herein are efficient strategies for overcoming immune rejection in cell-based transplantation therapies by creating generic donor stem cell lines.

Disclosed herein are stem cells in which the expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant factors is regulated compared to wild-type stem cells. Is.

Depending on the embodiment, one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C. Depending on the embodiment, the regulation of the expression of one or more MHC-I human leukocyte antigens comprises a decrease in the expression of one or more MHC-I human leukocyte antigens. In some embodiments, one or more MHC-I human leukocyte antigens have been removed from the cell's genome, thereby regulating the expression of one or more MHC-I human leukocyte antigens.

Depending on the embodiment, one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. Depending on the embodiment, the regulation of the expression of one or more MHC-II human leukocyte antigens comprises a decrease in the expression of one or more MHC-II human leukocyte antigens. In some embodiments, one or more indels were introduced into CIITA, thereby regulating the expression of one or more MHC-II human leukocyte antigens.

In some embodiments, the cells do not express HLA-A, HLA-B, and HLA-C. In certain embodiments, the cells are HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , and CIITA Indel ^/ Indel cells.

Depending on the embodiment, one or more tolerant factors are selected from the group consisting of HLA-G, PD-L1 and CD47. In certain embodiments, regulation of the expression of one or more tolerant factors comprises increased expression of one or more tolerant factors. In some embodiments, one or more tolerant factors are inserted at the AAVS1 safe harbor locus. In some embodiments, HLA-G, PD-L1 and CD47 are inserted at the AAVS1 safe harbor locus. In some embodiments, one or more tolerant factors inhibit immune rejection.

In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cells are hypoimmunogenic. In some embodiments, the stem cell is a human stem cell.

In some embodiments, the stem cells retain pluripotency. In some embodiments, the stem cells retain their ability to differentiate. In some embodiments, stem cells exhibit a reduced T cell response. In some embodiments, the stem cells exhibit protection from NK cell responses. In some embodiments, stem cells exhibit reduced macrophage phagocytosis.

Also disclosed herein are stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.

In some embodiments, the stem cells are HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , CIITA Indel ^/ Indel cells. In some embodiments, the stem cells express the tolerant factors HLA-G, PD-L1 and CD47. In some embodiments, the tolerant factor is inserted at the AAVS1 safe harbor locus. In certain embodiments, tolerant factors inhibit immune rejection.

In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cells are hypoimmunogenic.

Disclosed herein are methods of preparing hypoimmunogenic stem cells, which are MHC-I and MHC-II human leukocyte antigens of one or more stem cells and one or more tolerant sources of stem cells. It involves regulating the expression of sex factors compared to wild-type stem cells, thereby preparing hypoimmunogenic stem cells.

Depending on the embodiment, one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C. Depending on the embodiment, the regulation of the expression of one or more MHC-I human leukocyte antigens comprises a decrease in the expression of one or more MHC-I human leukocyte antigens. In some embodiments, one or more MHC-I human leukocyte antigens are removed from the stem cell genome, thereby regulating the expression of one or more MHC-I human leukocyte antigens.

Depending on the embodiment, one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. Depending on the embodiment, the regulation of the expression of one or more MHC-II human leukocyte antigens comprises a decrease in the expression of one or more MHC-II human leukocyte antigens. In some embodiments, one or more indels were introduced into CIITA, thereby regulating the expression of one or more MHC-II human leukocyte antigens.

In some embodiments, hypoimmunogenic stem cells do not express HLA-A, HLA-B, and HLA-C. In some embodiments, hypoimmunogenic stem cells are HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , and CIITA ^{indel / indel} cells.

Depending on the embodiment, one or more tolerant factors are selected from the group consisting of HLA-G, PD-L1 and CD47. Depending on the embodiment, regulation of the expression of one or more tolerant factors comprises increased expression of one or more tolerant factors. In some embodiments, one or more tolerant factors are inserted at the AAVS1 safe harbor locus. In some embodiments, HLA-G, PD-L1 and CD47 are inserted at the AAVS1 safe harbor locus. In some embodiments, one or more tolerant factors inhibit immune rejection.

In some embodiments, hypoimmunogenic stem cells retain pluripotency. In some embodiments, hypoimmunogenic stem cells retain their ability to differentiate. In some embodiments, hypoimmunogenic stem cells exhibit a reduced T cell response. In some embodiments, hypoimmunogenic stem cells exhibit protection from NK cell responses. In some embodiments, hypoimmunogenic stem cells exhibit reduced macrophage phagocytosis.

In some embodiments, stem cells are contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 1-2, thereby editing the HLA-A gene. Thus, the surface expression and / or activity of HLA-A in stem cells is reduced or eliminated. In some embodiments, stem cells are contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 3-4, thereby editing the HLA-B gene. , Reduces or eliminates surface expression and / or activity of HLA-B in stem cells. In some embodiments, stem cells are contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 5-6, thereby editing the HLA-C gene. , Reduces or eliminates surface expression and / or activity of HLA-C in stem cells. In some embodiments, the stem cell is contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and the ribonucleic acid having the sequence of SEQ ID NO: 7, thereby introducing the indel into CIITA and MHC-II in the stem cell. Decreases or eliminates surface expression and / or activity of human leukocyte antigens.

Also disclosed herein is a method for the preparation of hypoimmuneogenic stem cells, the method of which is one or more MHC-I and MHC-II human leukocyte antigens of stem cells and one or more tolerance. Containing that the expression of the progenitor factor is regulated relative to wild-type stem cells, thereby preparing hypoimmunogenic stem cells, the stem cells are categorized as Cas protein or a nucleic acid sequence encoding Cas protein, and SEQ ID NO: Contact with a first pair of ribonucleic acids having sequences 1-2, thereby editing the HLA-A gene to reduce or eliminate surface expression and / or activity of HLA-A in stem cells in the process. , Stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acid having the sequences of SEQ ID NOs: 3-4, thereby editing the HLA-B gene in the stem cells. A third of the ribonucleic acid having the Cas protein or the nucleic acid sequence encoding the Cas protein and the sequences of SEQ ID NOs: 5-6, in the process, which reduces or eliminates the surface expression and / or activity of HLA-B. Contact with a pair, thereby editing the HLA-C gene to reduce or eliminate surface expression and / or activity of HLA-C in stem cells, and during the process encode the stem cells, Cas protein or Cas protein. Contact with the protein sequence and the ribonucleic acid having the sequence of SEQ ID NO: 7, thereby introducing Indel into CIITA to reduce the surface expression and / or activity of the MHC-II human leukocyte antigen in stem cells or Exclude.

Also disclosed herein is a method of transplanting at least one hypoimmogenic stem cell into a patient, wherein the hypoimmunogenic stem cell is one or more MHC-I and MHC-. II The expression of human leukocyte antigen and one or more tolerant factors is regulated compared to wild-type stem cells.

Also disclosed herein are stem cells. In stem cells, the expression of MHC-I and MHC-II human leukocyte antigens may be decreased as compared with wild-type stem cells, and the expression of tolerable factors may be increased as compared with wild-type stem cells. , MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C, where the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. And here, the tolerant factor is CD47.

In some embodiments, reduced expression of MHC-I human leukocyte antigen comprises removing the MHC-I human leukocyte antigen from at least one allele of the cell. In some embodiments, reduced expression of MHC-II human leukocyte antigen comprises introducing one or more indels into CIITA. In some embodiments, stem cells further comprise reduced expression of CIITA. In some embodiments, the tolerant factor is inserted into the safe harbor locus of at least one allele of the cell.

In some embodiments, stem cells do not express HLA-A, HLA-B, and HLA-C. In some embodiments, stem cells do not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, stem cells do not express CIITA. In some embodiments, tolerant factors further include HLA-G and / or PD-L1.

Disclosed herein are stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, but do express CD47. In some embodiments, the cells are CIITA indel / indel, HLA-A-/-, HLA-B-/-, and HLA-C-/-stem cells.

Also disclosed herein is a method for preparing hypoimmunogenic stem cells. The method may include preparing hypoimmunogenic stem cells by reducing the expression of MHC-I and MHC-II human leukocyte antigens in the stem cells and increasing the expression of tolerant factors in the stem cells. Yes, in the method, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C, and in the method, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-. DR, as well as the tolerant factor in the method is CD47.

In some embodiments, reducing the expression of MHC-I human leukocyte antigen comprises removing the MHC-I human leukocyte antigen from at least one allele of stem cells. In some embodiments, reducing the expression of MHC-II human leukocyte antigen comprises introducing one or more indels into CIITA. In some embodiments, the method further comprises reducing the expression of CIITA. In some embodiments, increasing the expression of a tolerant factor comprises inserting the tolerant factor into the safe harbor locus of at least one allele of the cell.

In some embodiments, tolerant factors further include PD-L1 and / or HLA-G. In some embodiments, hypoimmunogenic stem cells do not express HLA-A, HLA-B, and HLA-C. In some embodiments, hypoimmunogenic stem cells do not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, hypoimmunogenic stem cells do not express CIITA.

The patent or application file contains at least one drawing made in color. A copy of this patent or publication of the patent application, including color drawings, is provided by requesting the Patent Office and paying the required fees.

We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1A presents a schematic diagram of the HLA-B and HLA-C CRISPR / Cas9 knockout strategies. Each scissors represents two sgRNAs. Purple, red, and green arrows indicate the primers used for PCR screening. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1B presents a schematic diagram of an HLA-A knockout strategy. Each scissors represents one sgRNA. Yellow arrows indicate the primers used for PCR screening. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1C presents a FACS contour diagram demonstrating the successful removal of HLA-A / B / C in HUES8. Wild-type (WT) or HLA-A / B / C knockout (KO) cells were treated with IFNγ for 48 hours and then stained with the indicated antibody. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1D shows a targeting strategy for the CIITA locus. Blue arrows indicate the primers used for PCR and Sanger sequencing. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1E shows the HLA-DR average fluorescence intensity (MFI) in EC of differentiated CD144 ⁺ WT and KO. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1F presents a schematic diagram illustrating the genotypes of WT, KO, KI-PHC, and KIPC cell lines. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1G shows a knock-in strategy for immunomodulatory molecules. Scissors represent sgRNAs that target the AAVS1 locus. Black and gray arrows indicate the primers used for PCR screening. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1H shows the expression of PD-L1 and HLA-G in KI-PHC cells. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1I shows the expression of CD47 in KI-PHC cells. The MFI compared to the WT cells is shown on the right side of the histogram. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 1J shows the expression of PD-L1 and CD47 in KI-PHC cells. Demonstrate that KO and KI cell lines retain pluripotency and potency. FIG. 2A shows immunofluorescence indicating that pluripotency markers were expressed by WT, KO, KI-PHC, and KI-PC human pluripotent stem cells (hPSC). The scale bar is 200 μm. Demonstrate that KO and KI cell lines retain pluripotency and potency. FIG. 2B shows that qRT-PCR was performed to investigate tribloodline markers after the hPSCs of WT, KO, KI-PHC, and KI-PC became three germ layers that were differentiated and displayed. show. Relative amounts were normalized relative to each gene level in undenatured hPSC. Demonstrate that KO and KI cell lines retain pluripotency and potency. FIG. 2C shows that G banding of chromosomes from KO, KI-PHC, and KI-PC cell lines demonstrated normal karyotype after successive stages of genomic manipulation. Demonstrate that KO and KI cell lines retain pluripotency and potency. FIG. 2D is a table showing PCR-based analysis of exon target external positions in modified hPSC strains. Demonstrate that KO and KI cell lines retain pluripotency and potency. FIG. 2E shows the results of target capture sequencing in% read combined with sequence changes at the external position of the target in the WT and modified hPSC strains. Black circle, SNP / polymorph (PM) site; red circle, edited external target position; blue circle, CIITA target mid-site as a positive control. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3A shows CD3 ⁺ (left panel, n = 8 donors), CD4 ⁺ (center panel, n = 6 donors), and CD8 ⁺ (right side) when co-cultured with WT, KO, or KI EC for 5 days. Panel, n = 6 donors) A scatter plot showing the percentage of T cell proliferation in a T cell population is presented. T cells cultured alone were used as a negative control. T cells activated with CD3 / CD28 beads play the role of a positive control. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^* P <0.05, ^** p <0.01. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3B shows CD69 ⁺ in the CD3 ⁺ (left panel), CD4 ⁺ (center panel), and CD8 ⁺ (right panel) T cell populations after co-cultured with WT, KO, or KI EC for 5 days. A scatter plot showing the percentage of cells (upper panel) and CD25 ⁺ cells (lower panel) is presented (n = 11 donors in all plots). The same negative and positive controls as in FIG. 3A were used. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^** p <0.01, ^*** p <0.001, ^*** p <0.0001. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3B shows CD69 ⁺ in the CD3 ⁺ (left panel), CD4 ⁺ (center panel), and CD8 ⁺ (right panel) T cell populations after co-cultured with WT, KO, or KI EC for 5 days. A scatter plot showing the percentage of cells (upper panel) and CD25 ⁺ cells (lower panel) is presented (n = 11 donors in all plots). The same negative and positive controls as in FIG. 3A were used. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^** p <0.01, ^*** p <0.001, ^*** p <0.0001. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3C presents a bar graph of IFNγ (left panel) and IL-10 (right panel) concentrations in medium after co-culturing WT, KO, or KI EC with CD3 ⁺ T cells from one representative donor. do. Spontaneous release from T cells alone was used as a negative control. Normal one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^** p <0.01, ^*** p <0.001. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3D presents a bar graph representing% cytotoxicity of T cells to EC of WT, KO, and KI (n = 6 donors). An LDH release assay was performed to calculate the cytotoxic percentage of T cells in each donor. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^* P <0.05, ^** p <0.01. Demonstrate a reduced T cell response to KO and KI cell lines in vivo. FIG. 4A presents a schematic diagram illustrating the workflow of allogeneic CD8 ⁺ T cell presensitization and T cell recall response assay in vivo. Demonstrate a reduced T cell response to KO and KI cell lines in vivo. FIG. 4B shows the increase in teratoma volume on days 5 and 7 after T cell injection as a percentage compared to day 0. Teratoma genotypes: WT (n = 9), KO (n = 7), KI-PHC (n = 6), and KI-PC (n = 7). Normal one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^* P <0.05. Demonstrate a reduced T cell response to KO and KI cell lines in vivo. FIG. 4C shows the increase in teratoma volume on day 0 of T cell injection as a percentage compared to 2 days before injection. Teratoma genotypes: WT (n = 9), KO (n = 7), KI-PHC (n = 6), and KI-PC (n = 7). Demonstrate a reduced T cell response to KO and KI cell lines in vivo. FIG. 4D shows WT (n = 8), KO (n = 7), KI-PHC (n = 6), and KI-PC (n = 7) teratomas collected 8 days after T cell injection. The relative mRNA expression of hCD8 (left panel) and IL-2 (right panel) is shown. Expression was normalized relative to RPLP0. Normal one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^* P <0.05, ^** p <0.01. Demonstrate a reduced T cell response to KO and KI cell lines in vivo. FIG. 4E shows representative hematoxylin and eosin (H & E) staining of WT, KO, KI-PHC, and KI-PC teratomas collected 8 days after T cell injection. Black arrows indicate T cell infiltration sites. Scale bar, 100 μm. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 5A presents a scatter plot of NK cell degranulation for VSMC of WT, KO, or KI-PHC (n = 7 donors). The percentage of NK cells to be degranulated was plotted as CD107a-expressing CD56 ⁺ % of cells for each donor. NK cells cultured alone were used as a negative control. NK cells treated with PMA / ionomycin served as a positive control. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^** p <0.01. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 5B presents a bar graph showing the percentage of NK cell damage to VSMC of WT, KO, and KI-PHC from one representative donor in the displayed effector / target (E / T) ratio (n). = Repeat 3 times). An LDH release assay was performed and NK cytolytic% was calculated as the specific lysis of NK cell killing VSMC to maximal cytolysis. Unpaired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard deviation. ^* P <0.05, ^*** p <0.001. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 5C presents a time-lapse plot of the macrophage phagocytosis assay (n = 5 monocyte donors). VSMCs of the displayed genotype were pretreated with staurosporine (STS) (right panel) or not (left panel) pHrodo red labeled and co-incubated with monocyte-derived macrophages for 6 hours. Images were taken every 20 minutes using the Celldiscover7 live cell imaging system. The integrated fluorescence intensity of pHrodo red + phagosome per image was analyzed. The data are mean ± standard error. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 5D presents a scatter plot of a macrophage phagocytosis assay in a 4-hour co-incubation (n = 9 monocyte donor, 3 independent experiments). The experimental conditions were the same as those in FIG. 5C. Paired one-way ANOVA followed by Tukey's multiple comparison test. The data are mean ± standard error. ^* P <0.05, ^** p <0.01. VSMC = vascular smooth muscle cells, NK = natural killer cells. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6A shows PCR support for HLA-B / -C knockouts using the primers shown in FIG. 1A. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6B shows PCR support for HLA-A knockouts using the primers shown in FIG. 1B. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6C shows a PCR product with primers flanking the CIITA cleavage site. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6D reveals that in the KO cell line, 1 bp (shown in red) was inserted into one CIITA allele and 12 bp (shown in dashed line) was removed from the other allele by the Sanger sequencing method. Is shown. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6E shows CD144 expression in differentiated WT and KO endothelial cells (EC). We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6F shows a workflow for generating ES cell lines of KO and KI. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 6G shows PCR support for knock-in of KI-PHC / KI-PC constructs using the primers shown in FIG. 1G. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7A shows the expression of CD47 in ES cells of WT and KI-PC. The MFI compared to WT cells is shown to the right of the histogram. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7B shows HLA-A2 expression in ES cells of WT, KI-PHC, and KI-PC after IFNγ treatment, supporting the removal of classical HLA class Ia molecules in KI cell lines. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7C shows the expression of CD144 in EC of differentiated WT, KI-PHC, and KI-PC. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7D shows the HLA-DR average fluorescence intensity (MFI), confirming the removal of HLA class II in the EC of differentiated KI-PHC and KI-PC. HLA-DR expression was analyzed on CD144 ⁺ cells. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7E shows the expression of CD140b in VSMC of differentiated WT, KO, KI-PHC, and KI-PC. We demonstrate that genome editing eliminates polymorphic HLA-A / -B / -C and HLA class II expression, as well as allowing expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7F presents a contour diagram (upper left panel) showing the expression of PD-L1 and HLA-G in VSMC of differentiated WT and KI-PHC. CD47 expression in VSMC of differentiated WT and KI-PHC (upper right panel). A contour map showing the expression of PD-L1 and CD47 in VSMC of differentiated WT and KI-PC (lower left panel). CD47 expression in VSMC of differentiated WT and KI-PC (lower right panel). We demonstrate that genome editing allows the elimination of polymorphic HLA-A / -B / -C and HLA class II expression, as well as the expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7G shows HLA-E expression upon IFN-γ stimulation in VSMCs of differentiated WT, B2M ^{− / −} , KO, and KI-PHC. Gray, isotype; colored, antibody. We demonstrate that genome editing allows the elimination of polymorphic HLA-A / -B / -C and HLA class II expression, as well as the expression of immunomodulatory factors from the AAVS1 safe harbor locus. FIG. 7H shows the relative expression of HLA-E mRNA in differentiated WT, B2M ^{− / −} , KO, and KI-PHC VSMC with or without IFN-γ stimulation. We present a sequenced chromatogram of the predicted exon target external position in the genetically modified hPSC strain and parent WT cells. We present a sequenced chromatogram of the predicted exon target external position in the genetically modified hPSC strain and parent WT cells. We present a sequenced chromatogram of the predicted exon target external position in the genetically modified hPSC strain and parent WT cells. We present a sequenced chromatogram of the predicted exon target external position in the genetically modified hPSC strain and parent WT cells. A sequence test from NGS is shown, which shows the edit at the target external position in the modified hPSC strain, and the SNP / polymorphic site observed in the modified strain as well as the WT cells. A sequence test from NGS is shown, which shows the edit at the target external position in the modified hPSC strain, and the SNP / polymorphic site observed in the modified strain as well as the WT cells. A sequence test from NGS is shown, which shows the edit at the target external position in the modified hPSC strain, and the SNP / polymorphic site observed in the modified strain as well as the WT cells. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines. FIG. 10A shows the gate opening and closing strategy used in the T cell proliferation and activation assay. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines. FIG. 10B presents one representative donor T cell proliferation assay using EC of WT, KO, and KI-PHC as target cells. CD3 ⁺ (upper panel), CD4 ⁺ (middle panel), and CD8 ⁺ (lower panel). T cells cultured alone were used as a negative control. T cells treated with CD3 / CD28 beads played a positive control role. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines. FIG. 10C shows doxycycline-induced PD-L1 expression in VSMC of WT. Demonstrate a decrease in T cell activity against KO and KI-PHC cell lines. FIG. 10D shows CD3 ⁺ (left panel), CD4 ⁺ (center panel), and CD8 ⁺ (right panel) T cells co-cultured with VSMC in the presence or absence of doxicyclin-induced PD-L1 expression. A scatter plot of percent T cell proliferation in the population is presented (n = 4 donors). T cells with reduced CFSE signal were quantified as proliferating cells. T cells cultured alone played the role of a negative control. T cells activated with CD3 / CD28 beads were used as positive controls. Paired two-sided t-test. The data are mean ± standard error. ^* P <0.05, ns, no significance. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 11A shows the expression of CD69 and PD-1 tested before and after priming one representative CD8 ⁺ T donor. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 11B presents a gate opening and closing strategy for the NK cell degranulation assay. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 11C presents a FACS contour diagram of an NK cell degranulation assay for one representative donor. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 11D shows the MFI of CD47 confirming the elimination of CD47 expression in differentiated CD47 ^{− / −} VSMC. Demonstrate that the KI cell line is protected from NK cells and macrophage responses. FIG. 11E presents a fluorescent image showing phagocytosis of VSMC prelabeled with pHrod red after 4 hours co-incubation with macrophages from one representative donor. The VSMC was either pretreated with staurosporine or left untreated. The image represents the superposition of the bright field and the red channel and highlights the fluorescent phagosome after masking with ZEN image analysis software. Scale bar, 200 μm. Demonstrate overcoming HLA barriers. FIG. 12A presents a schematic diagram of MHC class II and class I enhanceosomes. Targeting CIITA, the master regulator of MHC class II expression, blocks MHC class II expression. The promoter of the MHC class I gene is more complex, so removing NLRC5, a CIITA homolog that regulates MHC class I expression, only results in a decrease in MHC class I expression. Demonstrate overcoming HLA barriers. FIG. 12B shows a decrease in IFNg-induced MHC class I expression in NLRC5-/-CIITA- / hPSC. HUES9 cells of WT or displayed KO were stimulated with IFNg for 48 hours, subsequently stained for MHC class I expression and recorded on FACS. Removal of the accessory chain B2M completely blocks MHC class I surface expression, but makes these cells susceptible to NK cell killing. Demonstrate overcoming HLA barriers. FIG. 12C shows a targeted strategy for selectively removing the polymorphic HLA gene HLA-A / B / C from the hPSC genome. A schematic diagram of the target-oriented strategy is presented. Also shown is PCR support for each deletion in the HUES8 genome. Demonstrate a knock-in (KI) strategy that puts a tolerant factor into the safe harbor locus. FIG. 13A presents a schematic diagram of the KI construct. Demonstrate a knock-in (KI) strategy that puts a tolerant factor into the safe harbor locus. FIG. 13B presents a schematic diagram of the KI construct. Demonstrate a knock-in (KI) strategy that puts a tolerant factor into the safe harbor locus. FIG. 13C shows support for the deletion of HLA class I expression in two KI clones (C8 and C12). Demonstrate a knock-in (KI) strategy that puts a tolerant factor into the safe harbor locus. FIG. 13D shows that HLA-deficient KI clones C8 and C12 succeeded in overexpressing PD-L1 and CD47 from the AAVS1 safe harbor locus. Demonstrate a knock-in (KI) strategy that puts a tolerant factor into the safe harbor locus. FIG. 13E shows that the ultimate goal is to reverse the immunomodulatory activity of human trophoblast cells (high in PD-L1, HLA-G, CD47), where human trophoblast cells are pregnant. Induces resistance to semi-homogeneous fetuses (50% by parent and therefore outpatient). Demonstrate functional immune-silent cells for transplantation. FIG. 14A shows support for HLA expression in modified hPSC (HUES8). Deletion of MHC class I expression was supported by FACS in two independent HLA-A / B / C-/-CIITA-/-KO clones D1 and F2. Similar morphology of endothelial cells (EC) from KO clones was found. IFNγ-induced MHC class II expression in the expressed genotype EC demonstrates a deletion of HLA class II in HLA-A / B / C-/-CIITA-/-KO clones. Demonstrate functional immune silenced cells for transplantation. FIG. 14B shows the T cell proliferation assay (upper panel) and the NK cell degranulation assay (lower panel). For the cell proliferation assay (upper panel), CFSE-labeled T cell clones were used to evaluate T cell proliferation against EC derived from the displayed genotype HUES9. Deletion of the CFSE signal is proportional to T cell proliferation and represents the immunostimulatory activity of these cells. Although EC in WT causes predominant T cell proliferation over a 7-day period, T cell proliferation is reduced in the presence of two independent NLRC5-/-CIITA-/-KO clones, and B2M-/-. Not present when co-incubated with EC of CIITA − / − KO. For the NK cell degranulation assay (lower panel), HLA-deficient VSMCs (D1, F2) cause an improvement in NK cell degranulation compared to WT cells. PMA / ionomycin or HLA-deficient 221 cells were used as positive controls for NK degranulation. NC = negative control, NK cells alone. Demonstrate the generation of preclinical data. FIG. 15A shows improved engraftment of immunosilent human pluripotent stem cells in humanized mice. ES cells of the displayed genotype were transplanted into NSG mice reconstructed using the human immune system (BLT) to form teratomas. Teratoma dimensions and robustness were scored in a blinded manner 4-6 weeks after transplantation. Although WT teratomas show prominent features of rejection, the proliferation of NLCR5-/-CIITA-/-and B2M-/-CIITA-/-stem cells was found to be unrestricted and these were immunoprotected. Suggest that you are. Demonstrate the generation of preclinical data. FIG. 15B shows that cells can be removed by treatment with a CID dimer agent by introducing an inducible caspase-9 (iCasp9) killing switch. Frame picture of iCasp9 kill switch (left side). Dose escalation and time course of CID dimerizer in transiently transfected 293T cells (right). Eventually, the iCasp9 kill switch will be integrated into the safe harbor locus of modified immunosilent stem cells.

The invention disclosed herein uses genome editing techniques (eg, CRISPR / Cas or TALEN systems) to reduce or eliminate the expression of very important immune genes in stem cells, or in some cases,. A resistance-inducing factor is inserted, thereby making the stem cells and the differentiated cells prepared from them hypoimmunegenic and less susceptible to immune rejection by the subject to whom the cells are transplanted.

As used herein to characterize a cell, the term "hypoimmunogenic" generally means that the cell is less susceptible to immune rejection by the subject to whom the cell is transplanted. For example, compared to unmodified wild-type cells, such hypoimmunogenic cells have about 2.5%, 5%, 10%, 20%, 30 immune rejections by the subject to whom the cells are transplanted. %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more may be less susceptible. In some embodiments, genome editing techniques (eg, the CRISPR / Cas or TALEN system) are used to regulate (eg, reduce or eliminate) the expression of the MHC-I and MHC-II genes.

In certain embodiments, the invention disclosed herein relates to stem cells in which genomic modifications have reduced or removed a very important component of HLA expression. Similarly, in certain embodiments, the invention disclosed herein relates to stem cells into which one or more resistance-inducing factors have been inserted due to genomic modification. The present invention contemplates modifying a target polynucleotide sequence in any manner available to those of skill in the art, for example utilizing a TALEN, ZFN, or CRISPR / Cas system. Such CRISPR / Cas systems can use a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1 (6) e60). In some embodiments, the CRISPR / Cas system is a CRISPR type I system. In some embodiments, the CRISPR / Cas system is a CRISPR type II system. In some embodiments, the CRISPR / Cas system is a CRISPR V-type system. Of course, examples of methods utilizing CRISPR / Cas (eg, Cas9 and Cpf1) and TALEN are described in detail herein, but the invention is not limited to the use of such methods / systems. Other methods known to those of skill in the art for reducing or eliminating expression in target cells by targeting polynucleotide sequences can be utilized herein.

The present invention is intended to modify, eg, modify or cleave a target polynucleotide sequence in a cell for any purpose, in particular to reduce or eliminate expression or activity of the encoded product. It is intended to be modified to be. In some embodiments, the target polynucleotide sequence in the cell (eg, ES cell or iPSC) is modified to produce mutant cells. As used herein, a "mutant cell" generally refers to a cell whose resulting genotype is different from the genotype of origin or wild-type cell. In some cases, "mutant cells" exhibit a mutant phenotype, for example, when a normally functioning stem gene has been modified using the CRISPR / Cas system. In some embodiments, the target polynucleotide sequence in the cell is modified to correct or repair the genetic mutation (eg, restore the normal phenotype to the cell). In some embodiments, the target polynucleotide sequence in the cell is modified to induce a gene mutation (eg, disrupt the function of the gene or genomic sequence).

In some embodiments, the modification is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or combination thereof. As will be appreciated by those skilled in the art, indels in the translational region of the genomic sequence will result in frameshift mutations unless the indel length is a multiple of three. In some embodiments, the modification is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The CRISPR / Cas system can be used to induce indels or point mutations of any length in the target polynucleotide sequence.

In some embodiments, the modification results in a knockout of the target polynucleotide sequence or a portion thereof. For example, knocking out a target polynucleotide sequence in a cell can be done in vitro, in vivo or ex vivo for both therapeutic and research purposes. Knocking out a target polynucleotide sequence in a cell may be useful in the treatment or prevention of diseases associated with the expression of the target polynucleotide sequence (eg, knocking out a mutant allele of the cell with exvivo). By introducing cells with the knockout mutant allele into the subject).

As used herein, "knockout" involves deleting all or part of a target polynucleotide sequence in a manner that interferes with the function of the target polynucleotide sequence or its expression product.

In some embodiments, the modification results in reduced expression of the target polynucleotide sequence. The terms "decreased," "decreased," "decreased," and "decreased" are all used herein to generally mean a decrease in a statistically significant amount. However, for the avoidance of doubt, "decreased", "decreased", "decreased", and "decreased" are at least 10% reduction compared to the reference level, eg, at least about 20%, or at least about. 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% reduction, or 100% (ie, with a reference sample) Includes a reduction of up to 100%, including non-existent levels when compared, or any reduction between 10 and 100% when compared to a reference level.

The terms "increased", "increased", or "improved", or "activated" are all used herein to mean, generally, a statistically significant amount of increase. To. To avoid misunderstanding, "increased," "increased," or "improved," or "activated" is an increase of at least 10% when compared to a reference level, eg, when compared to a reference level. At least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%. Or an increase of up to 100%, including 100%, or any increase between 10 and 100%, or at least about 2 times, or at least about 3 times, or at least about 4 times when compared to reference levels. Or means an increase of at least about 5 times, or at least about 10 times, or any increase between 2 and 10 times or more.

The term "statistically significant" or "significantly" indicates statistical significance and generally means a concentration that is 2 standard deviations or less than the normal concentration of the marker. This term provides statistical evidence of differences. This is defined as the possibility of making a decision to reject the null hypothesis if the null hypothesis is actually correct. Judgments are made using the P-value.

In some embodiments, the modification is a homozygous modification. In some embodiments, the modification is a heterozygous modification.

In some embodiments, the modification results in the target polynucleotide sequence being modified from an undesired sequence to a desired sequence. The CRISPR / Cas system can be used to correct any type of mutation or error in the target polynucleotide sequence. For example, the CRISPR / Cas system can be used to insert a nucleotide sequence that is missing due to a deletion from the target polynucleotide sequence. The CRISPR / Cas system can also be used to remove or remove nucleotide sequences from insertion mutations from target polynucleotide sequences. In some cases, the CRISPR / Cas system can be used to replace the inaccurate nucleotide sequence with the correct nucleotide sequence (eg, to restore the function of the target polynucleotide sequence damaged by the loss-of-function mutation).

The CRISPR / Cas system can modify the target polynucleotide with surprisingly high efficiency. In certain embodiments, the modification efficiency is at least about 5%. In certain embodiments, the modification efficiency is at least about 10%. In certain embodiments, the modification efficiency is from about 10% to about 80%. In certain embodiments, the modification efficiency is from about 30% to about 80%. In certain embodiments, the modification efficiency is from about 50% to about 80%. In certain embodiments, the modification efficiency is about 80% or more. In certain embodiments, the modification efficiency is about 85% or higher. Depending on the embodiment, the modification efficiency is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more. Is. In some embodiments, the modification efficiency is equal to about 100%.

In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genome sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

In some embodiments, the CRISPR / Cas system is capable of directing the Cas protein or the nucleic acid sequence encoding the Cas protein and the Cas protein to the target motif of the target polynucleotide sequence and hybridizing to that motif. Includes two ribonucleic acids (eg, gRNA). In some embodiments, the CRISPR / Cas system is capable of directing the Cas protein or the nucleic acid sequence encoding the Cas protein and the Cas protein to the target motif of the target polynucleotide sequence and hybridizing to that motif. Includes nucleic acid or at least a pair of ribonucleic acids (eg, gRNA). As used herein, "protein" and "polypeptide" are used interchangeably to indicate a series of amino acid residues (ie, polymers of amino acids) linked by peptide bonds and modified amino acids (eg, polymers of amino acids). , Phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs are also included. Examples of polypeptides or proteins include gene products, native proteins, their homologs, paralogs, fragments and other equivalents, variants, and analogs.

In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprises a conservative amino acid substitution. In some cases, substitutions and / or modifications can prevent or reduce proteolysis of the polypeptide in the cell and / or prolong the half-life of the polypeptide. In some embodiments, the Cas protein can include peptide bond substitutions (eg, urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein can include naturally occurring amino acids. In some embodiments, the Cas protein can include alternative amino acids (eg, D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein can include modifications to include moieties (eg, PEGylation, glycosylation, lipidation, acetylation, terminal cap formation, etc.).

In some embodiments, the Cas protein comprises a core Cas protein. Examples of Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein is E. coli. E. coli subtype Cas protein (also known as CASS2) is included. E. Examples of E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Examples of Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises the Nmeni subtype Cas protein (also known as CASS4). Examples of Nmeni subtype Cas proteins include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Dvlug subtype Cas protein (also known as CASS1). Examples of Dvlug subtype Cas proteins include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Tneap subtype Cas protein (also known as CASS7). Examples of Tneap subtype Cas proteins include, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments, the Cas protein comprises the Hmari subtype Cas protein. Examples of Hmari subtype Cas proteins include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises an Apern subtype Cas protein (also known as CASS5). Examples of Apern subtype Cas proteins include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Mtube subtype Cas protein (also known as CASS6). Examples of Mtube subtype Cas proteins include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Examples of RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is Cas9 from any bacterial species or a functional portion thereof. The Cas9 protein is a member of the type II CRISPR system, which typically comprises transcoded small molecule RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas protein. The Cas9 protein (also known as the CRISPR-related endonuclease Cas9 / Csn1) is a polypeptide having an amino acid of 1368. Cas9 is complementary to two endonuclease domains, RuvC-like domains (residues 7-22, 759-766, and 982-989) that cleave target DNA that are not complementary to crRNA, and crRNA. It has an HNH nuclease domain (residues 810-872) that cleaves the target DNA.

In some embodiments, the Cas protein is a Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 from any bacterial species or a functional portion thereof. The Cpf1 protein is a member of the V-type CRISPR system. The Cpf1 protein is a polypeptide having about 1300 amino acids. Cpf1 has a RuvC-like endonuclease domain. Cpf1 uses a single ribonuclease domain to cleave the target DNA in the staggered pattern. Staggered DNA double strand disruption results in a 4 or 5-nt 5'overhang.

As used herein, a "functional moiety" retains the ability of a peptide to complex with at least one ribonucleic acid (eg, a guide RNA (gRNA)) to cleave a target polynucleotide sequence. Indicates a portion of the peptide. In some embodiments, the functional moiety comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional moiety comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex.

Not surprisingly, the present invention contemplates various ways in which a target polynucleotide sequence is contacted with a Cas protein (eg, Cas9). In some embodiments, the exogenous Cas protein can be introduced into cells in polypeptide form. In certain embodiments, the Cas protein can bind or fuse with a cell-permeable polypeptide or cell-permeable peptide. As used herein, "cell-permeable polypeptide" and "cell-permeable peptide" refer to a polypeptide or peptide that facilitates the uptake of a molecule into a cell, respectively. Cell-permeable polypeptides can have a detectable label.

In certain embodiments, the Cas protein can bind or fuse with a charged protein (eg, one having a positive charge, a negative charge, or an overall neutral charge). Such a bond may be a covalent bond. In some embodiments, the Cas protein can be fused with a large, positively charged GFP to significantly increase the ability of the Cas protein to permeate cells (Cronican et al. ACS Chem Biol. 2010; 5 (8). ): 747-52). In certain embodiments, the Cas protein can be fused with a protein transduction domain (PTD) to facilitate the entry of the Cas protein into cells. Examples of PTDs include Tat, oligoarginine and penetratin. In some embodiments, the Cas protein comprises a Cas polypeptide fused with a cell permeable peptide. In some embodiments, the Cas protein comprises a Cas polypeptide fused with PTD.

In some embodiments, the Cas protein can be introduced into cells containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (eg, Cas9 or Cpf1). The process of introducing nucleic acid into a cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection with viral vectors. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA (eg, a synthetic modified mRNA) as described herein.

In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one or two ribonucleic acids are introduced into the cell via viral transduction (eg, lentivirus transduction).

In some embodiments, the Cas protein complexes with one or two ribonucleic acids. In some embodiments, the Cas protein complexes with two ribonucleic acids. In some embodiments, the Cas protein complexes with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (eg, synthetically modified mRNA) as described herein.

The methods of the invention contemplate the use of any ribonucleic acid capable of directing the Cas protein to the target motif of the target polynucleotide sequence and hybridizing to that motif. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, the single ribonucleic acid comprises a guide RNA that directs the Cas protein to the target motif of the target polynucleotide sequence in the cell and hybridizes to that motif. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to the target motif of the target polynucleotide sequence in the cell and hybridizes to that motif. In some embodiments, both one or two ribonucleic acids include a guide RNA that directs the Cas protein to the target motif of the target polynucleotide sequence in the cell and hybridizes to that motif. The ribonucleic acid of the invention can be selected to hybridize to a wide variety of target motifs, depending on the particular CRISPR / Cas system used and the sequence of the target polynucleotide, as will be appreciated by those of skill in the art. One or two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids hybridize to a target motif having at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, one or two ribonucleic acids hybridize to a target motif having at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, one or two ribonucleic acids are designed to hybridize to a target motif that is directly adjacent to the deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, one or two ribonucleic acids are each designed to hybridize with a target motif that is directly adjacent to the deoxyribonucleic acid motif recognized by the Cas protein, and the deoxyribonucleic acid motif recognized by this Cas protein is targeted. Adjacent to the variant allele located between the motifs.

In some embodiments, at least one of one or two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7.

In some embodiments, at least one of one or two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7. In some embodiments, the at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7.

In some embodiments, each one or two ribonucleic acids comprises a guide RNA that directs the Cas protein to the target motif of the target polynucleotide sequence in the cell and hybridizes to that motif. In some embodiments, one or two ribonucleic acids (eg, guide RNAs) are complementary to and / or hybridize to sequences on the same strand of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (eg, guide RNAs) are complementary to and / or hybridize to sequences on opposite strands of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (eg, guide RNAs) are not complementary to and / or hybridize to the sequence on the opposite strand of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (eg, guide RNAs) are complementary to and / or hybridize with the overlapping target motifs of the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (eg, a guide RNA) are complementary to the offset target motif of the target polynucleotide sequence and / or hybridize to the rela motif.

In some embodiments, the target motif is a 17-23 nucleotide DNA sequence. In some embodiments, the target motif is at least 20 nucleotides in length. In some embodiments, the target motif is a 20 nucleotide DNA sequence.

In some embodiments, one or two ribonucleic acids hybridize to a target motif having at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, one or two ribonucleic acids hybridize to a target motif having at least one mismatch when compared to all other genomic nucleotide sequences in the cell. As one of ordinary skill in the art will admit, a variety of techniques can be used to select appropriate target motifs to minimize off-target effects (eg, bioinformatics analysis). In some embodiments, one or two ribonucleic acids are designed to hybridize to a target motif that is directly adjacent to the deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, one or two ribonucleic acids are each designed to hybridize with a target motif that is directly adjacent to the deoxyribonucleic acid motif recognized by the Cas protein, and the deoxyribonucleic acid motif recognized by this Cas protein is targeted. Adjacent to the variant allele located between the motifs.

In some embodiments, the target polynucleotide sequence in the cell uses a gene editing system (eg, TALEN, ZFN, CRISPR / Cas, etc.) to express one or more critical immune genes in the cell and / Or modified to reduce or eliminate activity. In some embodiments, the present disclosure removes a target polynucleotide sequence in a cell, a contiguous sequence of genomic DNA from one or both alleles of the cell (eg, one or more very important immune genes). It is provided to be modified (eg, using the CRISPR / Cas system) to be deleted). In some embodiments, the target polynucleotide sequence in the cell is modified to insert the gene mutation into one or both alleles of the cell (eg, using the CRISPR / Cas system). In yet another embodiment, the generic stem cells disclosed herein can be subjected to a complementary genomic editing approach (eg, using the CRISPR / Cas system), whereby the stem cell can be one of the cells or Deleting contiguous sequences of genomic DNA (eg, a very important immune gene) from both alleles, as well as one or more resistance inducers to one or both alleles of the cell (eg, HLA-G, CD47). And / or the insertion of PD-L1) are both modified to locally suppress the immune system and improve the engraftment of the implant.

Generic stem cells disclosed herein are used, for example, to diagnose, monitor, treat, and / or cure the presence or progression of a disease or condition (eg, type 1 diabetes or multiple sclerosis) in a subject. can do. As used herein, "subject" means human or animal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a young adult. In certain embodiments, the subject is treated in vivo, in vitro, and / or in utero. In certain embodiments, a subject in need of treatment according to the methods disclosed herein has, is suspected of developing, or is at high risk of developing the condition. In some embodiments, general purpose stem cells are transplanted into the subject.

Provided herein are novel cells, compositions, and methods that are useful in resolving such HLA-based immune rejection of transplanted cells.

Removal of MHC Class I and MHC Class II Genes In certain embodiments, the invention disclosed herein is one of the stem cell genomes that regulates the expression of MHC-I and / or MHC-II human leukocyte antigens. Concerning genomic modification of multiple target polynucleotide sequences. In some embodiments, a gene editing system is used to modify one or more target polynucleotide sequences. In some embodiments, the CRISPR / Cas system is used to remove one or more target polynucleotide sequences and / or introduce indels into one or more target polynucleotide sequences.

Efficient removal of the HLA barrier is by directly targeting polymorphic HLA alleles (HLA-A, -B, -C) and / or components of the MHC enhanceosome that are very important for HLA expression. It can be achieved by deleting (CIITA, etc.).

In certain embodiments, HLA expression is subject to interference. In some embodiments, HLA expression is a transcriptional regulator of HLA expression by targeting an individual HLA (eg, by knocking out HLA-A, HLA-B, and / or HLA-C expression). Interference is achieved by targeting (eg, CIITA). Depending on the embodiment, multiple HLAs can be targeted at the same time. For example, HLA-B and HLA-C are adjacent and can be targeted at the same time. In some embodiments, 95 kb deletion from the stem cell genome using CRISPR / Cas knocks out HLA-B and HLA-C, as well as the promoters of these two genes. In some embodiments, 13 kb deletion from the stem cell genome using CRISPR / Cas knocks out HLA-A and the promoter of this gene.

In certain embodiments, the stem cells disclosed herein are one or more human leukocyte antigens corresponding to MHC-I and / or MHC-II (eg, HLA-A, HLA-B, HLA-C). , HLA-DP, HLA-DQ, and / or HLA-DR) and are therefore characterized as hypoimmunogenic. For example, in certain embodiments, the stem cells disclosed herein are such that the stem cell or differentiated stem cell prepared from it does not express or exhibits reduced expression of one or more of the following MHC-I molecules: Have been modified to: HLA-A, HLA-B, and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C may be "knocked out" from the cell. Cells in which the HLA-A, HLA-B, and / or HLA-C genes have been knocked out may exhibit reduced or eliminated expression of each knocked out gene. In some embodiments, the stem cells disclosed herein are modified such that the stem cells or differentiated stem cells prepared from them do not express or exhibit reduced expression of one or more of the following MHC-II molecules: Yes: HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, one or more indels are inserted into a transcription regulator of HLA class II expression (eg, CIITA). Cells in which indels have been inserted into CIITA (eg, targeting exon 1) may exhibit reduced or eliminated expression of HLA-DP, HLA-DQ, and / or HLA-DR.

In some embodiments, the present disclosure has a genome in which the HLA-A gene has been edited to remove contiguous sequences of genomic DNA, thereby reducing surface expression of MHC class I molecules in the cell or population thereof. Or provide a stem cell that has been eliminated (eg, hypoimmogenic stem cells) or a population thereof. A contiguous sequence of genomic DNA refers to the cell or population thereof to the Cas protein or the nucleic acid encoding the Cas protein, and at least one ribonucleic acid or at least one pair of ribs selected from the group consisting of SEQ ID NOs: 1-2. It can be removed by contact with nucleic acid.

In certain embodiments, the present disclosure provides a method of modifying a target HLA-A sequence in a cell, wherein the HLA-A sequence is associated with a clustered repeat short sentence sequence repeat-associated (Cas) protein. , And contacting with at least one ribonucleic acid or at least one pair of ribonucleic acids, in which the ribonucleic acid directs the Cas protein to the target motif of the target HLA-A polynucleotide sequence and with this target. Hybridize, and during the method, the target HLA-A polynucleotide sequence is cleaved, and during the method, at least one ribonucleic acid or at least a pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1-2.

In some embodiments, the present disclosure has a genome in which the HLA-B gene has been edited to remove contiguous sequences of genomic DNA, thereby reducing surface expression of MHC class I molecules in the cell or population thereof. Or provide a stem cell that has been eliminated (eg, hypoimmogenic stem cells) or a population thereof. A contiguous sequence of genomic DNA refers to the cell or population thereof to the Cas protein or the nucleic acid encoding the Cas protein, and at least one ribonucleic acid or at least one pair of ribs selected from the group consisting of SEQ ID NOs: 3-4. It can be removed by contact with nucleic acid.

In certain embodiments, the present disclosure provides a method of modifying a target HLA-B sequence in a cell, wherein the HLA-B sequence is associated with a clustered repeat short sentence sequence repeat-associated (Cas) protein. , And contacting with at least one ribonucleic acid or at least one pair of ribonucleic acids, in which the ribonucleic acid directs the Cas protein to the target motif of the target HLA-B polynucleotide sequence and with this target. Hybridize, and during the method, the target HLA-B polynucleotide sequence is cleaved, and during the method, at least one ribonucleic acid or at least a pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3-4.

In some embodiments, the present disclosure has a genome in which the HLA-C gene has been edited to remove contiguous sequences of genomic DNA, thereby reducing surface expression of MHC class I molecules in the cell or population thereof. Or provide a stem cell that has been eliminated (eg, hypoimmogenic stem cells) or a population thereof. A contiguous sequence of genomic DNA refers to the cell or population thereof to the Cas protein or the nucleic acid encoding the Cas protein, and at least one ribonucleic acid or at least one pair of ribs selected from the group consisting of SEQ ID NOs: 5-6. It can be removed by contact with nucleic acid.

In certain embodiments, the present disclosure provides a method of modifying a target HLA-C sequence in a cell, wherein the HLA-C sequence is associated with a clustered repeat short sentence sequence repeat-associated (Cas) protein. , And contacting with at least one ribonucleic acid or at least one pair of ribonucleic acids, in which the ribonucleic acid directs the Cas protein to the target motif of the target HLA-C polynucleotide sequence and with this target. Hybridize, and during the method, the target HLA-C polynucleotide sequence is cleaved, and during the method, at least one ribonucleic acid or at least a pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 5-6.

In certain embodiments, the present disclosure comprises a genome in which the Class II transactivator (CIITA) gene has been edited and one or more indels have been introduced into exon 1, thereby in the cell or population thereof. Provides stem cells (eg, hypoimmogenic stem cells) or populations thereof in which surface expression of MHC class II molecules (eg, HLA-DP, HLA-DQ, and HLA-DR) is reduced or eliminated. One or more indels can be introduced by contacting the cell or population thereof with the Cas protein or the nucleic acid encoding the Cas protein and the ribonucleic acid consisting of SEQ ID NO: 7. In some embodiments, exon 1 of CIITA is the target of at least one ribonucleic acid or at least a pair of ribonucleic acids selected from the group consisting of the ribonucleic acid of SEQ ID NO: 7 and the group of SEQ ID NOs: 1-2.

In certain embodiments, the present disclosure provides a method of introducing one or more indels into a cell, wherein the CIITA sequence (eg, Exon 1 of CIITA) encodes a Cas protein or Cas protein. In the method, the ribonucleic acid directs the Cas protein to the target motif of the target CIITA polynucleotide sequence and hybridizes with this target, and comprises contacting with the nucleic acid and the ribonucleic acid, and one of the methods. Alternatively, multiple indels are introduced into exon 1 of the CIITA polynucleotide sequence, and in the method, the ribonucleic acid has the sequence of SEQ ID NO: 7. In some embodiments, exon 1 of CIITA is the target of at least one ribonucleic acid or at least a pair of ribonucleic acids selected from the group consisting of the ribonucleic acid of SEQ ID NO: 7 and the group of SEQ ID NOs: 1-2.

Insertion of Tolerability Factors In certain embodiments, one or more tolerant factors can be inserted or reinserted into a genome-edited stem cell line to create a generic donor stem cell with immunoprivilege. .. In certain embodiments, the generic stem cells disclosed herein are further modified to express one or more tolerant factors. Examples of tolerant factors include, without particular limitation, one or more of HLA-G, PD-L1 and CD47. Expression of such tolerant factors can inhibit immune rejection.

We use a genome editing system (such as the CRISPR / Cas-related homologous recombination repair (HDR) system) to actively inhibit immune rejection and use the safe harbor locus of tolerability factor (AAVS1). Has promoted insertion into loci). In some embodiments, the donor plasmid comprises an HLA-G expression cassette. In some embodiments, the donor plasmid comprises a PD-L1 expression cassette. In some embodiments, the donor plasmid comprises a CD47 expression cassette. In certain embodiments, the donor plasmid comprises a PD-L1, HLA-G, and CD47 expression cassette. In certain embodiments, the donor plasmid comprises a PD-L1 and CD47 expression cassette. The donor plasmid containing the expression cassette can target the AAVS1 locus of stem cells (eg, hypoimmunogenic stem cells). In certain embodiments, the donor plasmid can be used with ribonucleic acid to target the AAVS1 locus of hypoimmunogenic stem cells, in which case the ribonucleic acid has the sequence of SEQ ID NO: 8.

In some embodiments, the present disclosure provides a stem cell (eg, hypoimmunogenic stem cell) or a population thereof having a genome in which the stem cell genome is modified to express HLA-G. In some embodiments, the present disclosure provides a method of modifying a stem cell genome to express HLA-G. In certain embodiments, at least one ribonucleic acid or at least a pair of ribonucleic acids may be used to facilitate the insertion of HLA-G into a stem cell line.

In some embodiments, the present disclosure provides stem cells (eg, hypoimmunogenic stem cells) or populations thereof having a genome in which the stem cell genome is modified to express PD-L1. In some embodiments, the present disclosure provides a method of modifying the stem cell genome to express PD-L1. In certain embodiments, at least one ribonucleic acid or at least a pair of ribonucleic acids may be used to facilitate insertion of PD-L1 into a stem cell line.

In some embodiments, the present disclosure provides stem cells (eg, hypoimmunogenic stem cells) or populations thereof having a genome in which the stem cell genome is modified to express CD-47. In some embodiments, the present disclosure provides a method of modifying the stem cell genome to express CD-47. In certain embodiments, at least one ribonucleic acid or at least a pair of ribonucleic acids may be used to facilitate insertion of CD-47 into a stem cell line.

In some embodiments, the present disclosure discloses hypoimmogenic stem cells (eg, HLA-A, HLA-B, HLA) having a genome in which the stem cell genome is modified to express PD-L1, HLA-G, and CD47. -Stem cells modified to have C, HLA-DP, HLA-DQ, and HLA-DR expression removed) or a population thereof. In some embodiments, the present disclosure provides a method of modifying the stem cell genome to express PD-L1, HLA-G, and CD47.

In some embodiments, the present disclosure discloses hypoimmogenic stem cells (eg, HLA-A, HLA-B, HLA-C, HLA-) having a genome in which the stem cell genome is modified to express PD-L1 and CD47. Stem cells modified to have their expression of DP, HLA-DQ, and HLA-DR removed) or a population thereof. In some embodiments, the present disclosure provides a method of modifying the stem cell genome to express PD-L1 and CD47.

Generic Stem Cell In certain embodiments, the invention disclosed herein relates to generic stem cells. Generic stem cells may have reduced expression of one or more MHC-I and MHC-II human leukocyte antigens, as well as increased or excessive expression of one or more tolerant factors. be. In certain embodiments, the generic stem cells are HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , and CIITA Indel ^/ Indel cells, which cells are HLA-G, PD-. It exhibits increased expression of L1 and CD47.

In some embodiments, the stem cells described herein (eg, general purpose stem cells) exhibit one or more features. For example, stem cells retain their ability to differentiate, exhibit a reduced T cell response, exhibit protection from NK cell responses, and exhibit reduced macrophage phagocytosis.

General-purpose stem cells retain pluripotency, undergo tribloodline differentiation, and retain normal karyotype. For example, general purpose stem cells can retain expression of one or more of NANOG, OCT4, SSEA3, and TRA-1-60. In some embodiments, general purpose stem cells differentiate into three germ layers (eg, ectoderm, mesoderm, and endoderm) and maintain expression of all differentiation lineage markers.

In some embodiments, general purpose stem cells demonstrate a reduced T-cell adaptive immune response. For example, T cells (eg, CD4 ⁺ and CD8 ⁺ T cells) exhibit reduced priming and activation for general purpose stem cells. In addition, T cells exhibit reduced cytokine secretion to general purpose stem cells. Decreased expression of HLA-I and HLA-II molecules may result in decreased CD4 ⁺ and CD8 ⁺ T cell priming for general purpose cells. In some embodiments, expression of PD-L1 further suppresses activation of CD8 ⁺ T cells.

In some embodiments, general purpose stem cells are protected from NK cell rejection. Generic stem cells may be protected from NK cell rejection as a result of HLA-G expression. In some embodiments, general purpose stem cells exhibit reduced macrophage phagocytosis. Overexpression of CD47 and / or PD-L1 in general-purpose stem cells may minimize or inhibit macrophage phagocytosis of general-purpose stem cells.

Some Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application should have meaning generally understood by one of ordinary skill in the art. Furthermore, unless otherwise required by the context, the singular should include the plural and the plural should include the singular.

As used herein, the terms "comprising" or "comprises" are essential to the invention, the composition, the method, the kit, and their respective components (s). However, with respect to unspecified elements, whether they are mandatory or not, they still do not preclude their inclusion.

As used herein, the term "consisting essentially of" refers to the elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional properties (s) of the embodiments of the invention.

The term "consisting of" is such that the composition, method, kit, and components thereof are as described herein and are not listed in that description of the embodiment. Elements are also excluded.

Except as an example of operation or otherwise stated, all numbers representing the amounts of components or reaction conditions used herein are understood to be modified by the term "about" in all cases. Should be. The term "about" means ± 1% when used in terms of percentage.

The singular forms "a", "an", and "the" include multiple referents unless the context clearly indicates otherwise. Similarly, the word "or" shall include "and" unless the context clearly indicates otherwise. Further, of course, all base or amino acid dimensions given to a nucleic acid or polypeptide, as well as all molecular weight or molecular mass values, are approximations and are provided for illustration purposes. Suitable methods and materials, although similar or equivalent methods and materials as those described herein can be used in the practice or testing of the present disclosure, are described below. The term "comprises" means "includes". The abbreviation "eg (eg.)" Is derived from the Latin word expempli gratia and is used herein to indicate a non-restrictive illustration. That is, the abbreviation "for example (eg)" is synonymous with the term "for example".

The entire teachings of PCT Application No. PCT / US2016 / 031551, filing date May 9, 2016, are incorporated herein by reference. All other patents and publications identified are expressly herein for the purpose of explaining and disclosing, for example, the methods that may be used in connection with the present disclosure described in such publication. Incorporated as a medium reference. These publications are provided only because their disclosure is prior to the filing date of this application. In this regard, nothing should be construed as an endorsement that the inventors have no right prior to such disclosure, either on the grounds of prior inventions or for any other reason. With respect to these documents, any notice that they are on date or any expression that they are on date is based on the information available to the Applicants and what is the accuracy of the date or content of these documents. Does not configure approval.

Any of the various embodiments described and described herein, of course, to those skilled in the art, even if not within the scope already shown, are further modified and disclosed herein. The features shown in any of the other embodiments will be incorporated.

The following examples illustrate some embodiments and embodiments of the invention. As will be apparent to those skilled in the art, various modifications, additions, substitutions, etc. can be performed without altering the gist and scope of the invention, and such modifications and variants are described in the following patents. Included within the scope of the invention as defined in the claims. The following examples do not limit the invention in any way.

Treatments that utilize cells derived from human pluripotent stem cells for transplantation have the potential to revolutionize the way the disease is treated. A major obstacle to their clinical conversion is allogeneic cell rejection by the recipient's immune system. Strategies aimed at overcoming this immune barrier include cell bank construction of cells with established HLA haplotypes (Nakajima et al., 2007; Taylor et al., 2005) and patient-specific induced pluripotency. Generation of sex stem cells (iPSC) (Takahashi et al., 2007; Yu et al., 2007) can be mentioned. However, due to multiple constraints (de Rham and Village, 2014; Tapia and Scholar, 2016), such an approach cannot be widely applied and can be easily administered to any patient in need of a cellular product. The need for "instant delivery" cell preparations only stands out. Given that HLA class I molecules are expressed in virtually all nucleated cells as a first step in the manufacture of such general purpose stem cell formulations, we present cellular peptides for cytotoxic CD8 ⁺ T cells. It is necessary to remove HLA class I to prevent this. Moreover, removal of HLA class II should be considered. Because HLA class II is also highly polymorphic, it may be present upon IFNγ stimulation in certain hPSC-derived donor cell types, especially in specialized antigen-presenting cells (APCs) and endothelial cells (ECs). From (Ting and Forwardsdale, 2002). Recently, the power of the CRISPR / Cas9 genome editing system interferes with HLA class I expression in hPSCs or hematopoietic cells by knocking out the accessory chain beta-2-microglobulin (B2M) (Mandal et al., 2014; HLA Class II expression for and by targeting its transcriptional master regulator CIITA for Mattaplely et al., 2018; Meissner et al., 2014; Riolobos et al., 2013; Wang et al., 2015). (Chen et al., 2015; Mattapally et al., 2018) provided a tool for elimination. However, deletion of B2M also blocks the surface expression of the non-polymorphic non-classical HLA class Ib molecules HLA-E and HLA-G. HLA-E and HLA-G are required to maintain NK cell resistance (Ferreira et al., 2017; Lee et al., 1998b). Moreover, B2M-deficient cells are still known to be rejected by allogeneic CD8 ⁺ T cells (Glass et al., 1992). Therefore, individual deletion of the HLA-A / -B / -C gene may be a better strategy for protecting donor cells from CD8 ⁺ T cell cytotoxicity without losing HLA class Ib protective function. There is sex.

Another approach that has been studied to create "immediate delivery" cell formulations is the co-stimulation signal required for co-suppressive molecule expression and total T cell activation prior to HLA-T cell receptor (TCR) association. Blocking is mentioned. For example, ectopic expression of the T cell checkpoint inhibitors PD-L1 and CTLA-4Ig has been shown to protect stem cells from rejection in a humanized mouse model (Rong et al., 2014). Nevertheless, this approach leaves the HLA barrier intact and may result in hyperacute rejection of engraftment cells induced by existing anti-HLA antibodies (Iniotaki-Theodoraki, 2001; Masson et al., 2007). .. Moreover, CTLA-4Ig can also impair regulatory T cell (Treg) homeostasis and function, jeopardizing the establishment of post-transplant immune tolerance (Bour-Jordan et al., 2004; Salomon and Bluestone, 2001).

Innate immune cells, such as NK cells and macrophages, play an important role in priming adaptive immune responses in many situations, including chronic transplant rejection. A major concern associated with B2M removal is that this strategy makes donor cells vulnerable to NK cell killing because of "self-loss" (Raulet, 2006). More recently, Gornaluse et al. Expressed a B2M-HLA-E fusion construct in B2M-deficient cells to overcome NK cell lysis (Gornalusse et al., 2017). However, this approach cannot address NK cells lacking the inhibitory receptor NKG2A for HLA-E, and the reactivity of NK cells may remain of concern (Blaud et al., 1998a). Pegram et al., 2011). Therefore, HLA-G (Ferreira et al., 2017; Pazmany et al., 1996), which is an NK cell inhibitory ligand expressed at the maternal fetal border during pregnancy and acts through multiple inhibitory receptors, is an NK cell. It may be a better candidate to fully overcome the response. Moreover, macrophages, which contribute to the rejection of transplanted cells, may be regulated by the expression of CD47, a "don't eat me" signal that prevents the cells from being phagocytosed by macrophages (Chabra et). al., 2016; Jaiswal et al., 2009; Majeti et al., 2009). However, this approach has not yet been studied to protect hPSC and its differentiation derivatives from macrophage phagocytosis. Moreover, no compelling approach targeting both adaptive and innate immunity has yet been proposed.

Here we demonstrate that the CRISPR / Cas9 system can be used to selectively excise genes encoding polymorphic HLA class I members, HLA-A / -B / -C, from the hPSC genome. To. Moreover, the multiplexing capability of this system allows for simultaneous removal of HLA class II gene expression using a single guide RNA that targets CIITA. The resulting polymorphic HLA-deficient "immune-opaque" cells are further modified to target immunomodulatory factors PD-L1, HLA-G, and immune surveillance by T cells, NK cells, and macrophages, respectively. Expression of CD47 further attenuated the allo response in vitro and in vivo. The combination of these and other genetic modifications may ultimately result in a universal "quick delivery" cell formulation suitable for transplantation into any patient.

Results Given the high homology of the human MHC class I genes HLA-A, HLA-B, and HLA-C, which eliminate polymorphic HLA-A / -B / -C and HLA class II expression by genome editing, It has proved difficult to design specific short guide RNAs (sgRNAs) that target the translational regions of each gene using the CRISPR / Cas9 genome editing system. Therefore, a double-guided multiplex strategy targeting the untranslated regions adjacent to these genes was used to simultaneously excise these three from the genome of the hPSC strain (HUES8). At the HLA locus, HLA-B and HLA-C are adjacent, while HLA-A is closer to the telomere. Two sgRNAs were designed at sites upstream of HLA-B and downstream of HLA-C to simultaneously knock out adjacent HLA-B and HLA-C genes (FIG. 1A). The predicted 95 kb deletion also includes promoters for the two genes, which are defined as the H3K27Ac positive range in the UCSC Genome Browser. To knock out the entire HLA-A gene, one sgRNA was designed upstream of HLA-A and another sgRNA downstream of HLA-A (FIG. 1B). The expected 13 kb deletion includes the HLA-A promoter, according to the UCSC Genome Browser. Both deletions were confirmed by PCR amplicon straddling the predicted Cas9 cleavage site (FIGS. 6A-6B). It was verified by flow cytometry that the HLA-A / -B / -C protein was removed in the final HLA knockout clone (KO) (FIG. 1C).

Targeting CIITA, the master regulator of HLA class II expression, is well documented as a strategy to eliminate the three hyperpolymorphic HLA class II alleles, HLADP / -DQ / -DR, together. (Krawczik and Reith, 2006; Reith and Mach, 2001). SgRNAs targeting exon 1 of CIITA and having high cleavage efficiency have been previously reported (Fig. 1D) (Ding et al., 2013). This sgRNA was used in combination with an sgRNA targeting the HLA-A gene. The region spanning the cleavage site was amplified using a pair of PCR primers adjacent to the cleavage site in the first exon of CIITA. PCR amplicons were subjected to the Sanger sequencing method to identify both allelic frameshifts (FIGS. 6C-6D). To demonstrate that targeting CIITA resulted in a deletion of HLA class II expression, both WT and KO hPSCs were used using a previously published protocol (Patsch et al., 2015). Was differentiated into endothelial cells (EC). Notably, the differentiated WT and KO ECs expressed the EC marker CD144 (VE-cadherin) at equal levels, which did not affect the differentiation efficiency of the resulting cells by genome editing. This is shown (Fig. 6E). Importantly, the induction of HLA-DR expression upon IFNγ stimulation disappeared in the EC of KO (Fig. 1E). HPSC clones of KO with genotype HLA-A-/-HLA-B-/-HLAC-/-CIITA indel / indel were generated according to the workflow described in FIG. 6F. Taken together, these results demonstrate that multiple CRISPR / Cas9 genome editing can combine and highly specifically remove polymorphic HLA class I and II gene expression in hPSC.

It has been hypothesized that T-cell adaptive immune rejection can be eliminated by removing polymorphic HLA class Ia and class II molecules that knock in immunomodulatory factors into HLA knockout cell lines. However, HLA knockout cells still appear to be affected by innate immune cells involved in the allo-response, such as NK cells and macrophages, which are motivated and based on the following rationale: , The effect of introducing immunomodulatory factors was investigated: 1) Although the non-polymorphic HLA-E gene remains intact, its surface expression can be severely impaired by removal of the polymorphic HLA class I gene. It is highly potent because the major peptide presented by HLA-E is a leader peptide derived from other Class I molecules (Blaud et al., 1998b). That is, if any of the HLA class I other than HLA-E cannot be expressed, the donor cells may be vulnerable to NK cell lysis. Introducing HLA-G into HLA knockout cells was investigated to protect the modified cells from NK cells. 2) Macrophages are attracted to cytokines secreted at the engraftment site and are primed by antibody binding to phagocytose foreign cells. Since CD47 binds to the signal control protein alpha (SIRPa) on the surface of macrophages and acts as a signal "don't eat me", CD47 is significantly increased in certain tumors and tumors from macrophage phagocytosis. It has been well reported to help escape (Betancur et al., 2017; Jaiswal et al., 2009; Willingham et al., 2012; Zhao et al., 2016). Therefore, the goal was to overexpress CD47 in HLA knockout cells. 3) HLA-G can present a classical peptide derived from an intracellular protein to T cells (Diehl et al., 1996), which potentially reverts the cell line to CD8 + T cell immune surveillance. It seems to be exposed. Moreover, γδ T cells can directly recognize the antigen and elicit a cytotoxic response (Vantourout and Hayday, 2013). To counteract any residual T cell response, PD-L1 (Riley, 2009), a T cell checkpoint inhibitor that binds to the PD-1 receptor on activated T cells and directly suppresses T cell activity. I decided to knock in. Moreover, PD-L1 expression is transplanted by inhibiting PD-1 + NK cells (Beldi-Ferchiou et al., 2016; DellaChiesa et al., 2016) and PD-1 + macrophages (Gordon et al., 2017). It may also contribute to protecting cells from innate immune rejection.

Knock-in of immunomodulatory factors to the AAVS1 safe harbor locus was investigated to avoid disordered integration and placement effects on transgene expression (Sadelain et al., 2011). Two donor plasmids were designed. One has a PD-L1; HLA-G; CD47 expression cassette and the other has a PD-L1; CD47 expression cassette, both of which are CAGGS promoters with arms homologous to the AAVS1 locus on both sides. It was driven by (Fig. 1G). Donor plasmids were electroporated with sgRNA targeting the AAVS1 locus and introduced into HLA-A-/-HLAB-/-HLA-C-/-CIITA indel / indel clones. It was confirmed by PCR that the expression cassette was integrated into the AAVS1 locus (Fig. 6G). Two clones were isolated according to the workflow of FIG. 6F and analyzed by flow cytometry. The clones that expressed PD-L1 and HLA-G as compared to WT cells but did not significantly overexpress CD47 were named KI-PHC (Fig. 1H-1I) and expressed PD-L1. The second clone that showed elevated CD47 levels was named KI-PC (FIGS. 1J and 7A). Surface HLA-A2 levels were examined by flow cytometry on both KI clones to confirm removal of HLA class Ia (FIG. 7B). When hPSCs of KI-PHC and KI-PC were differentiated into CD144 + EC (Fig. 7C), it was observed that there was no HLA-DR expression by flow cytometry after IFNγ stimulation (Fig. 7D). That is, the immunomodulatory factor was successfully inserted into the AAVS1 safe harbor locus of HLA class Ia and II null cells. In summary, three modified hPSC strains, KO, KI-PHC, and KI-PC (Fig. 1F) were generated.

Next, confirmation of transgene expression and HLA-E expression in the derivative of the modified hPSC strain was examined. For this purpose, the modified hPSC was differentiated into vascular smooth muscle cells (VSMC). VSMCs of WT, KO, KI-PHC, and KI-PC expressed the VSMC marker CD140b (PDGFRB) at equal levels, confirming similar differentiation efficiencies (FIG. 7E). In VSMC of KI-PHC, a subpopulation with slightly higher expression of PD-L1 and HLA-G was observed as compared with VSMC of WT, but in the large population, the levels of PD-L1 and HLA-G were significantly increased. (Fig. 7F). However, no increase in CD47 expression was observed in VSMC of KI-PHC (Fig. 7F). This may be the result of incomplete expression from the target-oriented cassette. In the target-oriented cassette, all three gene products are linked by 2A-peptides (Fig. 1G). Similarly, in VSMC of KI-PC, a small subpopulation with slightly higher levels of PD-L1 and CD47 compared to VSMC of WT and a large population with higher levels were observed (FIG. 7F).

When VSMC of WT was stimulated with IFNγ, they dramatically increased HLA-E surface expression. In contrast, cell surface HLA-E protein levels were significantly reduced in KO VSMCs (FIG. 7G), not because HLA-E gene expression was impaired in KO VSMCs (FIG. 7G). FIG. 7H). Surprisingly, the surface HLA-E expression of VSMC of KI-PHC was not restored by HLA-G expression (Fig. 7G). Nevertheless, HLA-G surface transport was undamaged in VSMC of KI-PHC (Fig. 7F). This further motivated the introduction of this tolerant factor into modified cell products to compensate for the reduced HLA-E surface expression in the HLA-A / -B / -C null background.

KO and KI cell lines retain pluripotency and differentiation potential Expression of NANOG, Oct4, SSEA3, SSEA4, and TRA-1-60 to assess whether the modified hPSC strain retained pluripotency. , KO, KI-PHC, and KI-PC hPSCs were evaluated by immunofluorescence and found to be equivalent to those of unmodified hPSCs (FIG. 2A). In addition, hPSCs of KO, KI-PHC, and KI-PC differentiated into three germ layers. The expression of ectoderm, mesoderm, and endoderm markers was tested by qRT-PCR and compared to unmodified hPSC-derived trigerm. All of the differentiated lineage markers analyzed were found to be expressed in each germ layer cell from these hPSCs (FIG. 2B). In addition, hPSCs of KO, KI-PHC, and KI-PC showed normal karyotype (Fig. 2C). That is, despite multiple gene modification rounds, these modified hPSC strains maintained pluripotency, undergoed tribloodline differentiation, and retained normal karyotype.

To analyze the potential off-target effects of sgRNA used in the manipulation of the hPSC strain, the top 21 computer-predicted exon target external positions were PCR amplified from the modified hPSC strain and the parent WT hPSC. No undesired edits were found at these sites except for the pseudogene HLA-H (HFE) from the Sanger sequencing of the PCR product, whereas the pseudogene HLA-H (HFE) was found in the genome to be HLA-. It was shown to be a perfect match for the upstream sgRNA of HLA-A used to remove A (FIGS. 2D and 8). More extensively, target capture sequencing was performed for all 648 predicted target external positions of the eight sgRNAs used in this study. After enrichment with a specially designed RNA bait, enriched DNA fragments were sequenced by next-generation sequencing (NGS) for each predicted target external position. Sequence reads from each cell line were aligned and compared to the hg38 genomic reference sequence to calculate the percentage of reads that had been modified. As a result, HLA-H (HFE) was identified as off-target in all three cell lines, in addition to the 12 identified natural SNPs / polymorphic sites. Moreover, intron targeting external positions derived from targeting HLA-C were detected in TRAF3 in all three cell lines, and as a result of AAVS1 sgRNA in CPNE5 in the KI-PC cell line, intron targeting external. Positions were detected (FIGS. 2E and 9). In summary, although three off-target events were detected, the modified hPSC strains retained pluripotency and their ability to differentiate into all three germ layer cells as well as VSMC and EC, respectively, on the WT counter. It was maintained with the same differentiation efficiency as the part.

Decreased T-cell response to KO and KI cell lines Given that elimination of polymorphic HLA class Ia expression is expected to eliminate T-cell adaptive immune responses, then with modified cell lines We examined investigating the activity of T cells in co-cultures. In addition to KO cells, KI-PHC cells were also used to determine whether expression of the T cell checkpoint inhibitor PD-L1 further suppressed T cell activity. Four separate in vitro T cell immunoassays were performed: T cell proliferation assay, activation assay, cytokine secretion assay, and killing assay. Since HLAI expression is moderate in hPSC (de Almeida et al., 2013; Drukker et al., 2002), modified hPSC and WT hPSC, followed by IFNγ stimulation, EC expressing both HLAI and II. Differentiated into VSMC expressing only HLAI, and then used for each immunoassay.

ECs from WT, KO, and KI-PHC were pretreated with IFNγ for 48 hours for a T cell proliferation assay, followed by co-cultured with CFSE-labeled allogeneic CD3 + T cells for 5 days. T cells were then stained for CD3 / 4/8 and analyzed for dilution of the CFSE signal by flow cytometry as a reading of T cell proliferation in different T cell subpopulations (FIG. 10A). A FACS plot of one representative T cell donor is shown in FIG. 10B. As expected, the percentage of total proliferating T cells (CD3 +) was KO EC (4.17% ± 0.89%) compared to incubation with WT EC (8.29% ± 1.23% SEM). Decreased when incubated with SEM) or EC of KI-PHC (3.87% ± 0.73% SEM) (FIG. 3A, left panel). CD4 + T cells follow a similar pattern, with WT EC (5.03% ± 0.89% SEM) being KO EC (3.58% ± 0.86% SEM) or KI-PHC EC (3.49). % ± 0.83% SEM) induced more CD4 + T cell proliferation (FIG. 3A, central panel). Moreover, CD8 + cytotoxic T cells are KO EC (7.71% ± 1.89% SEM) or KI-PHC when compared to WT EC (14.32% ± 2.39% SEM). When co-cultured with EC (5.95% ± 1.48% SEM), there was a significant reduction in proliferation (FIG. 3A, right panel). Importantly, the proliferation of CD8 + T cells was significantly less in the presence of KI-PHC EC when compared to the co-culture of KO with EC (FIG. 3A, right panel). This indicates that CD8 + T cell activation was further suppressed by overexpression of PD-L1 in the HLA null background. To further investigate the inhibitory role of PD-L1 in the response of different T cell subpopulations, an inducible PD-L1 expressing hPSC strain was generated, differentiated into EC, and then subjected to a T cell proliferation assay. It was found that only with CD8 +, T cell proliferation was reduced in the presence of PD-L1 expressed EC and not with CD4 + when compared to the EC of WT. This argues for the specific inhibitory effect of PD-L1 on the CD8 + T cell subset (FIGS. 10C-10D).

The same co-culture of T cells and EC as target cells was used to test the expression of T cell activation markers CD25 and CD69 (FIG. 3B). KI-PHC EC (4.91% ± 0) when compared to T cells co-incubated with WT EC (6.43% ± 0.71% SEM; 9.30% ± 1.51% SEM) In a co-culture with .74% SEM; 5.04% ± 1.24% SEM) or KO EC (5.12% ± 0.77% SEM; 5.40% ± 1.29% SEM). A decrease in the percentage of CD25 + and CD69 + T cells (CD3 +) was observed (FIG. 3B). The same tendency was observed in the CD4 + and CD8 + cell populations (FIG. 3B). Also found was that when co-cultured with WT EC, an increase in the percentage of CD25 + cells was observed in the CD4 + cell population, while an increase in the percentage of CD69 + cells was observed in the CD8 + cell population. However, no significant reduction in T cell activation marker expression was observed for KI-PHC EC when compared to KO EC.

Next, the levels of the T cell effector cytokines IFNγ and IL-10 secreted into the medium were tested over a 5-day T cell-EC co-culture process. Compared to the IFNγ and IL-10 levels (4747 ± 556.1SD; 54.56 ± 17.22SD) observed in the medium after contact with the EC of WT, the levels of both cytokines KO T cells. It was lower in the medium when contacted with either (3214 ± 180.5SD; 5.09 ± 0.16SD) or KI-PHC EC (2635 ± 132.9SD; 3.56 ± 0.63SD). .. This indicates that cytokines secreted from T cells were reduced relative to the KO or KI-PHC cell line (FIG. 3C).

To quantify T cell killing, lactate dehydrogenase (LDH) released from VSMC was measured as an alternative indicator of T cell cytotoxicity. Given that VSMC expresses only HLAI, in this situation only CD8 + T cells were expected to be activated by HLAI-TCR association. Cytotoxicity of CD8 + T cells to VSMC of KI-PHC (15.31%) when compared to KO (18.86% ± 4.34% SEM) and WT (37.65% ± 7.64% SEM) VSMC. ± 4.52% SEM) was found to be the lowest (Fig. 3D). This observation suggests that the cytotoxicity of CD8 + T cells was further suppressed by PD-L1 of VSMC of KI-PHC, which is consistent with the results of the CD8 + T cell proliferation assay. Taken together, observations from the T cell immunoassay demonstrate that CD4 + and CD8 + T cell priming for KO and KI-PHC cell lines is reduced as a result of removal of HLAI and II molecules. CD8 + T cell activation was further suppressed by expression of PD-L1 in the KIPHC cell line.

To evaluate T cell response in vivo, WT and modified hPSC were subcutaneously transplanted into immunodeficient mice to form teratomas over a course of 4-6 weeks. Presensitized allogeneic CD8 + T cells were then transplanted as adopted cells by tail vein injection and teratoma growth was monitored for an additional 8 days (FIG. 4A). T cells used for injection were activated (CD69 +) and showed no signs of post-sensitization depletion (PD-1 +) as measured by CD69 and PD-1 expression of CD8 + T cells before and after priming (PD-1 +). FIG. 11A). Consistent with the assumption that only WT cells would be rejected, 7 days after injection of CD8 + T cells, WT teratomas showed a slower volume increase than KO teratomas. This was not due to the slow growth rate of the WT teratoma itself (FIGS. 4B-4C). These results suggest that KO teratomas were protected from T-cell rejection. Moreover, although not significant, the average volume of KI-PHC and KI-PC teratomas was also larger than that of WT teratomas 7 days after injection of T cells (FIG. 4B). Also, both KO and KI cell line-derived teratomas of T cell infiltration, as supported by qPCR of the human effector T cell markers CD8 and IL-2 (FIG. 4D) and histology (FIG. 4E). Showed a decrease. Taken together, these observations suggest that removal of polymorphic HLA molecules from the cell surface of transplanted cells can effectively block T-cell rejection in vivo, which is an in vitro observation. Consistent with the result.

KI cell lines are protected from NK cell and macrophage responses Deletion of HLAIA molecules and damage to HLA-E surface expression indicate that hPSCs of KO and their derivatives are vulnerable to NK cell lysis. As expected, on the other hand, the KI-PHC cell line should be protected from NK cellular rejection as a result of HLA-G expression. To test this hypothesis, allogeneic NK cells were isolated from healthy donors and incubated with VSMC of WT, KO, or KI-PHC. CD56 + NK cells were analyzed by flow cytometry for surface expression of the degranulation marker CD107a as a reading of NK cell activation (FIG. 11B). Notably, NK cell degranulation in the presence of VSMC in KO was not significantly higher than in the presence of VSMC in WT (10.16% ± 2.96% SEM) (10.16% ± 2.96% SEM). FIG. 5A), which suggests a deletion of the NK cell activation signal in the hPSC-derived VSMC. However, in agreement with the hypothesis, the percentage of CD107a + degranulated NK cells (5.43% ± 0.95% SEM) in the co-culture of KI-PHC with VSMC was in the presence of KO VSMC (13). It was found to be significantly lower than .51% ± 2.51% SEM) (Fig. 5A). This suggests that NK cell activity is actually inhibited by HLA-G expression in VSMC of KI-PHC. A FACS plot of one representative donor is shown in FIG. 11C. LDH released from apoptotic VSMC after co-incubation with NK cells was also tested to quantify the cytotoxicity of NK cells. Consistent with NK cell degranulation, it was observed that when NK cells were incubated with KI-PHC VMSC, the cytotoxicity of NK cells was reduced (FIG. 5B).

Finally, macrophage activity was tested with a pH sensitive fluorescent dye (pHrod red) that signals the phagocytosis of phagocytic cells. It was hypothesized that macrophage phagocytosis was reduced by overexpression of the macrophage "don't eat me" signal CD47 in a derivative of the modified hPSC cell line. Considering that no significant increase in CD47 expression was observed in VSMC of KI-PHC (FIG. 7F), VSMC differentiated from the KI-PC cell line was used for these assays. This VSMC showed a much higher CD47 level than the VSMC of WT (Fig. 7F). In addition, a CD47 knockout (CD47 − / −) cell line was generated as a positive control for macrophage phagocytosis, and flow cytometry confirmed a deletion of CD47 cell surface expression (FIG. 11D). VSMCs differentiated from WT, CD47 − / −, and KI-PC cells are labeled pHrod red and treated with staurosporine (STS) to induce apoptosis or remain untreated. And then incubated with allogeneic macrophages isolated from healthy donors. The appearance of the red signal, which is an index of VSMC phagocytosed by macrophages, was monitored by live cell imaging, and the fluorescence intensity was quantified. Notably, the VSMC of KI-PC, with or without STS treatment, showed a significant reduction in macrophage phagocytosis when compared to VSMC of CD47 − / − or WT (FIG. 5C). -FIG. 5D and FIG. 11E). Although these data cannot rule out the possibility that PD-L1 expressed in VSMC of KI-PC also contributes to the inhibition of macrophage phagocytosis, overexpression of CD47 actually results in the modified hPSC-derived VSMC. Demonstrate that macrophage phagocytosis may be minimized (Fig. 7F).

Discussion In this study, multiple CRISPR / Cas9 genome editing was applied to knock out the highly polymorphic HLA-A / -B / -C gene and to target the CIITA gene of hPSC to express the HLA class II gene. Succeeded in blocking. We also introduced immunomodulatory factors PD-L1, HLA-G, and CD47 into the AAVS1 locus using CRISPR / Cas9-related homologous recombination repair (HDR). Modified hPSC derivatives were found to show significantly lower induced immune activation as well as killing by T and NK cells and minimal phagocytosis by macrophages.

An approach that eliminates HLA class Ia expression specifically excises HLA-A / -B / -C of the polymorphic HLA class Ia gene, while the gene B2M and the non-polymorphic HLA class Ib gene HLA-E, -F and -G were left intact. The resulting 95 kb deletion included MIR6891 and four pseudogenes as well as the HLA-B / -C gene, but no change was observed in the growth rate or differentiation efficiency of the KO or KI cell line. Interestingly, for unknown reasons, HLA-E surface expression was not restored by HLA-G expression in KI-PHC cells, which means that the leader peptide from HLA-G was transported by HLA-E surface. It was inconsistent with previous reports (Lee et al., 1998a) that it was sufficient for promotion.

The HLA knockout (KO) hPSC strain was generated by genome editing using 7 different sgRNAs. The hPSCs of KI-PHC and KI-PC are clones derived from the KO strain, which were edited with additional sgRNAs targeting the AAVS1 locus. Of the 648 predicted target external positions for the 8 sgRNAs used, the exon off-target event was transcribed as a result of one of the sgRNAs used to remove HLA-A from the genome, the pseudogene HLA-. Only one was observed in H. When translated, the observed 2bp deletion found in both alleles appears to result in a mutation that causes a frameshift. Mutations in HLA-H have been observed to be associated with hereditary hemochromatosis, a rare iron storage disorder (Feder et al., 1996), but the observed HLA-H (HFE) mutations. Did not affect the growth rate or differentiation efficiency of the cell types tested in this study. Of note, this is either by designing a different sgRNA or by identifying clones that do not carry this particular off-target mutation by identifying the genotype of the HLA-H locus. There is a possibility that untargeted events can be avoided. Moreover, the use of sgRNA / Cas9 ribonuclear protein complexes (RNPs) to direct targets allows for transient editing over plasmid-based approaches (Roth et al., 2018). This should reduce the number of untargeted events per cell line and should therefore be adopted in the future.

As expected, removal of polymorphic HLA expression in hPSCs and their derivatives, such as EC and VSMC, resulted in reduced T cell response in vitro and in vivo. An interesting observation obtained from the T cell assay was that overexpression of the checkpoint inhibitor PD-L1 had a significant effect only on CD8 + T cell proliferation and cytotoxicity. There may be multiple explanations for this: 1) PD-L1 receptor, PD-1 levels are higher in CD8 + T cells than in CD4 + T cells. 2) CD8 + T cells are the most responsive cell type to contact with target cells in this assay, and thus negative regulator PD-1 will also be expressed at higher levels. It should be noted that neither explanation is contradictory to each other, as the strength of T cell activation and PD-1 expression are associated in a negative feedback loop (Riley, 2009). Moreover, it was found that PD-L1 alone had an effect on CD8 + T cell proliferation even in the absence of HLA. This suggests that PD-L1 can act as a tolerant factor even in the absence of proliferative HLA-TCR interactions. Also, background T cell activity was observed in the T cell activation assay and cytokine secretion assay, even in co-cultures with the KIPHC cell line when compared to negative controls. This may be due to experimental settings, given that target cells may secrete factors that promote T cell activation, regardless of the presence of HLA.

Although acute transplant rejection is predominantly T-cell, the role of other immune cells, such as macrophages, NK cells, and B cells, must also be considered for engraftment and long-term survival of therapeutic cells. .. The NK cell assay suggests that HLA-G expression was able to control NK cell activity. Moreover, overexpression of CD47 effectively reduced macrophage phagocytosis. Nevertheless, as recently reported, PD-L1 was co-expressed in both KI cell lines, which included PD-1 + NK cells (Beldi-Ferciou et al., 2016; DellaChiesa et al., 2016) and PD. It can also affect the activity of -1 + macrophages (Gordon et al., 2017), which may contribute to the observed phenotype. For long-term engraftment, in particular, antibody-dependent cellular cytotoxicity (ADCC) by NK cells and allogeneic antibody-dependent complement activation as a major driver of chronic transplant rejection must be investigated (Baldwin et al). ., 2016; Djamari et al., 2014; Michaels et al., 2003). Additional introduction of factors known to inhibit ADCC and complement activation, such as CD59 (Meri et al., 1990), may allow durable engraftment. , Can be assumed. Ultimately, in vivo experiments will help clarify the degree of protection that modified cells may have after transplantation. While there are various humanized mouse models so far, they have limitations in reproducing a complete human immune response. That is, the development of an improved in vivo model for testing cell transplantation and rejection may be required (Brehm et al., 2014; Li et al., 2018; Melkus et al., 2006; Longvaux. et al., 2014).

Overcoming the immune barrier to transplantation not only overcomes the allobarrier, but also an autoimmune disease in which certain cell types are attacked by the patient's own immune system and need to be replaced, such as type 1 diabetes (T1D). And it is expected to provide an exciting new method that may treat multiple sclerosis and the like. That is, the generation of general-purpose cells that can be safely transplanted to anyone is expected to bring out all the possibilities of regenerative medicine.

Experimental procedure CRISPR gRNA sequence HLA-A upstream: 5'-GCCGCCTCCCCACTTGCGCT-3'(SEQ ID NO: 1)
HLA-A downstream: 5'-CACATGCAGCCCACGAGCCG-3'(SEQ ID NO: 2)
HLA-B upstream _1: 5'-ATCCCTAAATATGGTGTCCC-3'(SEQ ID NO: 3)
HLA-B upstream _2: 5'-TCCCTAAAATATGGTGTCCCT-3'(SEQ ID NO: 4)
HLA-C downstream _1: 5'-GTGATCCGGGGTATGGGCAGT-3'(SEQ ID NO: 5)
HLA-C downstream _2: 5'-TGATCCGGGTATGGGCAGTG-3'(SEQ ID NO: 6)
CIITA: 5'-TCCATCTGGGTCATAGAAG-3' (SEQ ID NO: 7)
gRNA_AAVS1-T2: 5'-GGGGCCACTAGGGACAGGAT-3'(SEQ ID NO: 8)

PCR and qPCR probes / primers PCR primers used in Figure 6:
Purple_F: 5'-CACTCAGAGCAAAGGTCAGATG-3'(SEQ ID NO: 9)
Purple_R: 5'-AGACTTGAATCCATAAGCCCAA-3'(SEQ ID NO: 10)
Red_F: 5'-GACAAGTCTCGGGAGAGGGTTT-3'(SEQ ID NO: 11)
Red_R: 5'-AGACTTGAATCCATAAGCCCAA-3'(SEQ ID NO: 12)
Green_F: 5'-CACTCAGAGCAAAGGTCAGATG-3' (SEQ ID NO: 13)
Green_R: 5'-TTTGTTGTCAGCAGACATAGG-3'(SEQ ID NO: 14)
Yellow_F: 5'-CTGGTTATTCCCCATTCTG-3'(SEQ ID NO: 15)
Yellow_R: 5'-AAGCATTCACTCCCTGACCCTG-3'(SEQ ID NO: 16)
Blue_F: 5'-GTCTTCCTCCCAGGCAGCTCA-3'(SEQ ID NO: 17)
Blue_R: 5'-TGAGGGGTGGGGGATACCGGA-3'(SEQ ID NO: 18)
Black_F: 5'-TCGACCTACCTCTTCCGCA-3'(SEQ ID NO: 19)
Black_R: 5'-TAGGGGGGCGTACTTGCATA-3'(SEQ ID NO: 20)
Gray_F: 5'-CCGTTCTCCTGTGGATTCGG-3'(SEQ ID NO: 21)
Gray_R: 5'-TCTCTGGCTCCATCGTAAGC-3'(SEQ ID NO: 22)
PCR primers used in FIG. 8:
HLA-F-AS1_F: 5'-GTCGCTTCAGTCAGGACACA-3'(SEQ ID NO: 23)
HLA-F-AS1_R: 5'-GAAGGTGCTGTTTGGCACAG-3'(SEQ ID NO: 24)
ITGA6_F: 5'-CCTTCAACTTGGACACTCGGG-3'(SEQ ID NO: 25)
ITGA6_R: 5'-CCACGGGCCAACTACC-3'(SEQ ID NO: 26)
HEATR1_F: 5'-TTACCCAGTTCATATCAGCCA-3'(SEQ ID NO: 27)
HEATR1_R: 5'-AGGGGTAAGCTGCAAACTTCTT-3'(SEQ ID NO: 28)
PTDSS2_F: 5'-GACCTCCACAGGGACTAGGT-3'(SEQ ID NO: 29)
PTDSS2_R: 5'-TTTGGAGTTGGTGCTCCCTC-3'(SEQ ID NO: 30)
CTBS_F: 5'-GCCCTCATCGAGTGGTCAAA-3'(SEQ ID NO: 31)
CTBS_R: 5'-CCGCTAGACCTCTGCTTG-3'(SEQ ID NO: 32)
ACSBG1_F: 5'-CTGGGTGTCAATTGATGGCGT-3'(SEQ ID NO: 33)
ACSBG1_R: 5'-GCCACATCTAAAAGGCAGCG-3'(SEQ ID NO: 34)
AC07882.1_F: 5'-GTTTGTGGGTGCTGGTCAAC-3'(SEQ ID NO: 35)
AC07885.2._R: 5'-CTAGGCAACAGTGACAGGG-3'(SEQ ID NO: 36)
HIDK4_F: 5'-GGACCATCATGTCGGAGACC-3'(SEQ ID NO: 37)
HIDK4_R: 5'-GACCTGGGAGTCACACGAAC-3' (SEQ ID NO: 38)
ACSBG1_F: 5'-CTGGGTGTCAATTGATGGCGT-3'(SEQ ID NO: 39)
ACSBG1_R: 5'-GCCACATCTAAAAGGCAGCG-3'(SEQ ID NO: 40)
HIC2_F: 5'-AAGTGTTCGGTCTGGCGAGAA-3'(SEQ ID NO: 41)
HIC2_R: 5'-GCTCTGCTTGGTACGGACTG-3'(SEQ ID NO: 42)
HLA-H_F: 5'-AGGTGATGTATGGCTGCGAC-3'(SEQ ID NO: 43)
HLA-H_R: 5'-TCCTTCCCGTTCTACGAGTA-3'(SEQ ID NO: 44)
HLA-K_F: 5'-GGTATGAACAGCAGCCAAC-3'(SEQ ID NO: 45)
HLA-K_R: 5'-GCGTCTTGTGTTCCCTGGTA-3'(SEQ ID NO: 46)
HLA-G_F: 5'-ACCCTACTACTGGGAGAACC-3'(SEQ ID NO: 47)
HLA-G_R: 5'-AGGCTCTCCTTTGTTCAGCC-3'(SEQ ID NO: 48)
PYCRL_F: 5'-CCTAGCCACGTGTGACTCAA-3' (SEQ ID NO: 49)
PYCRL_R: 5'-TGCCGTCCCAGTACAATC-3'(SEQ ID NO: 50)
RAB11FIP4_F: 5'-CGAGGGAGGGCAAAATTGAGT-3'(SEQ ID NO: 51)
RAB11FIP4_R: 5'-GAAGAAGGGACAAGGGGTGG-3'(SEQ ID NO: 52)
CHFR_F: 5'-GAGCTTTGAGCAGAGTGTTA-3'(SEQ ID NO: 53)
CHFR_R: 5'-CTGGGAGCATGCATTTGTGAGA-3'(SEQ ID NO: 54)
PNCK_F: 5'-CTGTTGGCAGGTGAACCCT-3'(SEQ ID NO: 55)
PNCK_R: 5'-CTGGGAAGGCTTGTTCTCTG-3'(SEQ ID NO: 56)
AMN_F: 5'-AGAGCTCAAGGTCCCAAGTG-3'(SEQ ID NO: 57)
AMN_R: 5'-GGGTAACTCACTCGGAGGTC-3'(SEQ ID NO: 58)
FUT1_F: 5'-TGGATTTCCAGAACCCCATCC-3'(SEQ ID NO: 59)
FUT1_R: 5'-GGGAACTCTCCTCTTGGTCT-3'(SEQ ID NO: 60)
NPPA_F: 5'-GAGCTTCTGCATTGGTCCCT-3'(SEQ ID NO: 61)
NPPA_R: 5'-TCTGATCCGATCTGCCCTCCCT-3'(SEQ ID NO: 62)
SYBR-based qPCR primer:
AFP_F: 5'-AAATGCGTTTTCGTTGCTTT-3'(SEQ ID NO: 63)
AFP_R: 5'-GCCACAGGCCAAATAGTTTGT-3'(SEQ ID NO: 64)
SOX17_F: 5'-CTCTGCCTCCTCACCACGAA-3'(SEQ ID NO: 65)
SOX17_R: 5'-CAGAATCCAGACCTGCACAA-3'(SEQ ID NO: 66)
BRACHYURY_F: 5'-AATTGGTCAGCCTTGGAAT-3'(SEQ ID NO: 67)
BRACHYURY_R: 5'-CGTTCGCTCACAGACCACA-3'(SEQ ID NO: 68)
FLK1_F: 5'-TGATCGGAAATGACACTGGA-3' (SEQ ID NO: 69)
FLK1_R: 5'-CAGACTCCATGTTGGTCAC-3'(SEQ ID NO: 70)
MAP2_F: 5'-CAGGTGCGCGAGACTGTGAAAATTGAGAGTG-3'(SEQ ID NO: 71)
MAP2_R: 5'-CACGCTGGATCTGCCTGGGGACTGTG-3'(SEQ ID NO: 72)
PAX6_F: 5'-GTCCATTTTTGCTTGGAAA-3' (SEQ ID NO: 73)
PAX6_R: 5'-TAGCCAGGTTGCAAGAAACT-3'(SEQ ID NO: 74)
TaqMan gene expression assay:
HLA-E: Hs03045171_m1
CD8: Hs00233520_m1
IL-2: Hs00174114_m1
RPLP0 (internal standard): Hs9999999902_m1

FACS antibody α-HLA-A2 (PE-bound type), clone BB7.2, BioLegend, catalog number 343305
α-HLA-ABC (PE-bound type), clone W6 / 32, BioLegend, catalog number 311406
α-HLA-E (PE-bound type), clone 3D12, BioLegend, catalog number 342603
α-HLA-G (PE-bound type), clone MEM-G / 9, Abcam, catalog number ab24384
α-HLA-DR (APC-bound type), clone MEM-12, Thermo Fisher Scientific, catalog number MA1-1347
α-B2M (APC-bound type), clone 2M2, BioLegend, catalog number 316311
α-PD-L1 (APC-bound type), clone 29E. 2A3, BioLegend, Catalog No. 329708
α-PD-1 (APC-bound type), clone EH12.2H7, BioLegend, catalog number 329908
α-CD3 (APC-bound), clone UCHT1, BioLegend, catalog number 300412
α-CD3 (Pacific Blue ™ bound type), clone UCHT1, BioLegend, catalog number 300418
α-CD4 (PE / Cy7 binding type), clone RPA-T4, BioLegend, catalog number 300511
α-CD8 (PE-bound type), clone SK1, BioLegend, catalog number 344705
α-CD25 (Alexa Fluor® 700-binding type), clone MA251, BioLegend, catalog number 356117
α-CD47 (PE-bound type), clone CC2C6, BioLegend, catalog number 323108
α-CD56 (PE binding type), clone HCD56, BioLegend, catalog number 318306
α-CD69 (Alexa Fluor® 647 binding type), clone FN50, BioLegend, catalog number 310918
α-CD107a (APC-bound), clone H4A3, BioLegend, catalog number 328620
α-CD144 (PE-bound type), clone 55-7H1, BD Biosciences, catalog number 560410

Isotype Isotype 1: Mouse IgG2b, κ isotype control (APC-bound), BioLegend, Catalog No. 400322
Isotype 2: Mouse IgG2b, κ isotype control (PE-bound), BioLegend, Catalog No. 401208
Isotype 3: Mouse IgG2a, κ isotype control (PE-bound), BioLegend, Catalog No. 400214

Immunofluorescence antibody α-OCT4, Abcam, Catalog number ab19857
α-NANOG, Abcam, catalog number ab21624
α-SSEA3, Millipore, Catalog number MAB4303
α-SSEA4, Millipore, catalog number MAB4304
α-TRA-1-60, Millipore, Catalog number MAB4360
Donkey Anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor® 488 Complex, Life Technologies, Catalog No. A-21206
Donkey anti-mouse IgG (H + L) secondary antibody, Alexa Fluor® 488 complex, Life Technologies, Catalog No. A-21202
Donkey anti-mouse IgM heavy chain secondary antibody, Alexa Fluor® 555 complex, Life Technologies, Catalog No. A-21426

Human ES cell culture, electrical perforation, and drug selection HUES8 cells (Cowan et al., 2004) were grown on plates pre-coated with Geltrex (Life Technologies) and supplemented with penicillin / streptomycin in mTeSR1 (StemCell Technologies). Was cultured in. For passage, cells were dissociated for 5-10 minutes using the GentleCell Dissociation Reagent (StemCell Technologies) and reseeded in fresh medium supplemented with RevtitaCell ™ (Thermo Fisher Scientific). Due to electroporation, as previously described (Peters et al., 2013), HUES8 cells were dissociated into single cells, and for gene knockout, 50 μg of pCas9_GFP (Addgene #) in 10 million cells. 44719) and electroporation with a total of 50 μg of gRNA plasmid. To knock in the gene at the AAVS1 locus, cells were electroporated with 50 μg pCas9_GFP, 25 μg gRNA_AAVS1-T2 (Addgene # 41818), and 40 μg double-stranded donor plasmid. For the purpose of gene knockout, cells were collected 48 hours after electroporation. GFP-expressing cells were concentrated by FACS (FACSAria II, BD Biosciences) and reseeded into 10 cm tissue culture plates in fresh medium supplemented with Revita Cell ™ at 15,000 cells / plate to generate single cell colonies. Formed. Alternatively, for gene knock-in, 48 hours after electroporation, cells were screened for 5 days with 2 μg / ml Blasticidin (Thermo Fisher Scientific). The cell colonies were then manually selected and grown.

CRISPR / Cas9 Genome Editing 500 base pairs in each region upstream or downstream of HLA-A / B / C were amplified from HUES8 or HEK293T cells and subjected to Sanger sequencing (Genewiz). The sequence conserved between the two cell lines was selected as the reference sequence, and the CRISPR design tool (available from: crispr.mit.edu) and CCTop (Stemmer et al., Available below) developed by the FengZhang laboratory of MIT. 2015) was used to design the sgRNA. The sgRNAs at the top of the ranking were carefully selected and cloned into a gRNA expression vector (Addgene # 41824). The gRNA plasmid was then transfected into HEK293T cells, genomic DNA was extracted, and PCR amplicon covering the cleavage site was analyzed by TIDE (available below: tide.nki.nl) for targeted medium efficiency. The single guide RNA with the highest targeted medium activity was used for genome editing of HUES8 cells. To construct a knock-in donor plasmid, the ORFs of PD-L1, HLA-G, and CD47 were individually cloned and ligated by 2A sequences using Gibson Assembly® (New England BioLabs). Then, after cleavage of Flpe-ERT2, a 3-in-1 cassette was inserted between the Sal I and Mlu I restriction sites in the AAVS1-Blastsaidin-CAG-Flpe-ERT2 plasmid (Addgene # 68461). Details of genome editing in human ESC have been previously described (Peters et al., 2013).

Generation of HLA Knockout (KO) Cell Lines Briefly, to knock out adjacent HLA-B / -C genes, a total of 4 sgRNAs, along with Cas9 expression plasmids, in wild-type (WT) HUES8 Simultaneous electric drilling. Homozygous knockout clones were then screened using the primers shown in FIG. 6A (HLA-B / -C ^-/- efficiency: 1.56%). Heterozygous knockout clones were also observed (HLA-B / -C ^+/- efficiency: 7.8%). Homozygous clones were further validated by RT-PCR for removal of HLA-B / -C mRNA expression, and normal karyotype was confirmed by nCounter human karyotype assay (data not shown). Finally, one normal clone of the case was selected to further target the HLA-A and CIITA genes in a single electroporation. HLA-A knockout clones were screened by PCR using the primers shown in FIG. 6B and flow cytometry using the α-HLA-A2 antibody. The primers and sanger sequencing shown in FIG. 6E were performed to identify CIITA knockout clones. As a result, only heterozygous clones (HLA-A ^+/- CIITA ^{+ /} Indel) were observed after the first round targeting HLAA / CIITA (HLA-A ^+/- efficiency: 3.68%). .. Therefore, another round of electroporation with HLA-A / CIITA sgRNA was applied to one normal heterozygous clone of the case and the same screening strategy was adopted. Eventually, one homozygous clone (HLA-A ^{− / −} CIITA Indel ^/ Indel) was generated, but from FACS analysis this clone was a mixed clone and still 1% HLA-A ⁺ cells. It became clear that he was holding. After subcloning, pure homozygous clones (HLA knockout, KO) were obtained.

Karyotype analysis Karyotype G banding was performed by Cell Line Genetics.

Induction of differentiation into three germ layers WT and gene-edited HuES8 cell lines are differentiated into ectoderm, mesoderm, and endoderm according to a protocol based on the monolayer of the STEMdiff ™ Trihematology Differentiation Kit (StemCell Technologies). rice field.

Differentiation into endothelial cells and vascular smooth muscle cells Human endothelial cells (EC) and vascular smooth muscle cells (VSMC) were differentiated according to a published protocol (Patch et al., 2015). Briefly, for EC differentiation, ESCs were seeded in N2B27 medium supplemented with 8 uM CHIR99021 (Cayman Chemical) and 25 ng / ml BMP4 (Peprotech) for 3 days to induce lateral mesoderm. The medium was then replaced with StemPro-34 supplemented with 200 ng / ml VEGF (Peprotech) and 2 μM forskolin (Abcam) to induce EC for 2 days. The cells were then enriched with CD144 ⁺ cells using MACS cell isolation (Miltenyi Biotec). CD144 ⁺ cells were seeded on EBM ™ -2 supplemented with EGM ™ -2 BulletKit ™ (Lonza) on a fibronectin (Corning) -coated plate and further differentiated for at least 7 days. To differentiate into VSMC, ESC was inoculated in the same medium as for EC differentiation for 3 days. On days 4 and 5, the medium was replaced with N2B27 supplemented with 12.5 ng / ml PDGF-BB (Peprotech) and 12.5 ng / ml activin A (Cell Guidance Systems). From day 6 onwards, cells were dissociated and seeded in a gelatin-coated dish supplemented with a smooth muscle growth supplement (Thermo Fisher Scientific) in medium 231 for further differentiation.

Isolation and Culture of Human Primary Immune Cells Blood from a healthy anonymous donor (Leukopack) was obtained from the Jackson Transfusion Center at Massachusetts General Hospital (Boston). Negative selection kits (RosetteSep ™ human T cell enrichment cocktail, RosetteSep ™ human NK cell enrichment cocktail, and RosetteSep ™ human monocyte enrichment, respectively, with human primary T cells, NK cells, or CD14 ⁺ monocytes. Isolated by cocktail, StemCell Technologies). The isolated T cells were cultured in X-VIVO 10 (Lonza) medium. Medium supplemented with 5% human AB serum (Valley Biomedical), 5% fetal bovine serum, 1% penicillin / streptomycin, GlutaMAX, MEM non-essential amino acids (ThermoFisher Scientific), and 20 U / ml IL-2 (Peprotech). bottom. The isolated NK cells were cultured in RPMI 1640 containing L-glutamine (Corning). RPMI 1640 was supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The isolated monocytes were differentiated into macrophages in RPMI 1640. RPMI 1640h was supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and 25-50 ng / ml M-CSF (Peprotech).

Flow cytometry PBS containing 1% fetal bovine serum (FBS) was used as wash buffer and stain buffer. PBS containing 4% FBS was used as block buffer. For EC and VSMC, FcR blocking reagent (Miltenyi Biotec) was added to the blocking buffer at a 1: 1000 dilution. Briefly, immune cells or other dissociated single cells are washed once and placed on ice for 20 minutes. Blocked with block buffer. Cells were stained with antibody for 30-60 minutes on ice, washed twice and then analyzed with FACSCalibur ™ or LSR II (BD Biosciences). Data were plotted using FlowJo software (BD).

When using the In vitro T cell proliferation assay VSMC, cells were first treated with mitomycin (Fisher Scientific). 100,000 EC or VSMCs were seeded in 24-well plates and treated with IFNγ (100 ng / ml) for 48 hours before assaying. On day 0 of co-incubation, isolated CD3 + T cells were labeled with CellTrace ™ CFSE (Thermo Fisher Scientific) according to the instruction manual. Adherent EC or VSMC was washed twice with PBS and then co-incubated with 500 k CFSE-labeled T cells in T cell medium supplemented with 20 U / ml IL-2 for 5 days. T cells were then stained with anti-CD3 / 4/8 antibody and then analyzed for CFSE intensity with LSR II. T cells cultured for 5 days without target cells were used as a negative control. T cells treated with Dynabeads ™ human T activator CD3 / CD28 beads (Thermo Fisher Scientific) for 5 days served as a positive control.

In vitro T cell activation assay and cytokine secretion assay ESC-derived EC was used as the target cell. The conditions for co-culture were the same as for the T cell proliferation assay, with the exception that T cells were not labeled. After co-culture for 5 days, T cells were stained for T cell activation markers and then analyzed with LSR II. For the quantification of multiple secretory cytokines, supernatants were collected and analyzed by a special MSD U-PLEX Platform (Meso Scale Discovery) according to the instruction manual. T cells or target cells cultured for 5 days were used as a negative control. T cells activated with Dynabeads ™ human T activator CD3 / CD28 beads (Thermo Fisher Scientific) for 5 days served as a positive control. Background activation was assessed using T cells incubated in EC or VSMC conditioned medium. The conditioned medium was prepared as described above.

In vitro T / NK cell killing assay ESC-derived VSMC was used as the target cell. For the T cell killing assay, the co-incubation conditions were the same as for the T cell activation assay. For the NK cell killing assay, 40K VSMC and NK cells were co-incubated in 96-well U bottom in 200 μl NK cell medium for 20 hours at the indicated effector / target ratios and then the supernatant was collected. After co-incubation, supernatants were collected and analyzed with the Pierce ™ LDH Cell Injury Assay Kit (Thermo Fisher Scientific) according to the instructions. T cell medium or NK cell medium (RPMI-10) was used as a background control. Single-cultured T / NK cells or single-cultured target cells were used as controls for spontaneous LDH release. Lysis target cells at the endpoint were used for maximum LDH release.

Presensitization of allogeneic human CD8 + T cells Human primary CD8 ⁺ T cells have been isolated using RosetteSep ™ human CD8 ⁺ T cell enrichment cocktails and previously described using HUES8 derived germ layers. Pre-sensitized as shown (Gornaluse et al., 2017). Briefly, germ layers were induced in suspension for 5 days, followed by adhering culture for an additional 4 days. CD8 ⁺ T cells were then co-cultured with attached germ layer cells for presensitization. During this process, extracellular matrix derived from heterologous material sources such as gelatin was avoided to prevent non-specific T cell activation.

In vivo T Cell Recall Response Assay All animal experiments were performed according to the rules of the Harvard International Animal Care and Use Committee. No randomization was used. All procedures were performed in a blinded manner. 8-10 week old male immunodeficient SCID Beige mice (Taconic) were used for teratoma formation. Two million HUES8 cells were encapsulated in a blood clot, and the blood clot was inserted subcutaneously into each flank of SCID Beige mice. After the teratoma became palpable, the size of the teratoma was measured weekly with a caliper. Four to six weeks after hESC transplantation, one million pre-sensitized allogeneic human CD8 ⁺ T cells were injected into mice through the tail vein. After T cell injection, the dimensions of the teratoma were measured on days 2, 5, and 7. Teratoma dimensions were also measured 2 days prior to T cell injection. On day 8 post-injection, teratomas were collected and analyzed by qPCR and hematoxylin and eosin (H & E) staining. In this study, two allogeneic CD8 ⁺ T cell donors were used under the same experimental conditions and the results were combined. The histological examination was performed by the Harvard Stem Cell Research Institute's tissue examination center facility.

In vitro NK cell degranulation assay 300,000 adherent ESC-derived VSMCs were seeded in 24-well plates 24 hours prior to the assay. The next day, VSMCs were washed once with PBS and then co-incubated in NK cell medium with 100 K freshly isolated NK cells. The NK cell medium was supplemented with α-CD107a APC (Biolegend) and eBioscience ™ protein transport inhibitor cocktail (Thermo Fisher Scientific). After adding the NK cells to the wells, the plates were sunk at 2,000 rpm for 5 minutes to achieve adequate contact between the effector and the target. After 20 hours of co-incubation, NK cells were stained with α-CD56 PE (Biolegend) and then analyzed for CD107a cell surface expression with FACSCalibur ™. NK cell cultures containing no target cells were used as a negative control. NK cells treated with a cell activation cocktail (without Breferdin A) were used as a positive control for degranulation. The cell activation cocktail contains PMA (12-myristic acid-13-phorbol acetate) and ionomycin.

In vitro macrophage phagocytosis assay RosetteSep ™ human monocyte enriched cocktails (StemCell Technologies) were used to isolate monocytes from donor blood by negative selection. Monocytes are seeded in serum-free medium for attachment and supplemented with 10% FBS, 1% penicillin / streptomycin, and 25 ng / ml M-CSF (Peprotech) for 1-3 weeks to mature into macrophages. Was sown in RPMI 1640. Two days prior to the assay, macrophages were reseeded on 96-well μ plates (ibidi) at a density of 100 K / well. For the assay, differentiated VSMCs were pretreated with 200 nM staurosporine (Sigma) for 1.5 hours for use in the "STS treated" group. The VSMC was dissociated and labeled with pHrodo red (IncuCite) at 37 ° C. for 1 hour. 30,000 labeled VSMCs were added to each of the macrophage-containing wells and the co-incubated cultures were immediately transferred to the Celldiscover 7 live cell imaging platform (Zeiss). Images of red fluorescence emitted when phagocytic cells were phagocytosed were taken every 20 minutes, one per well for 6 hours. For each image, the integrated intensity (average fluorescence intensity ^* total area) was analyzed using ZEN image processing software (Zeiss). pHrodo red ⁺ particles indicate phagosomes within macrophages that have phagocytosed VSMC.

Generation of CD47-/-and B2M-/-HUES8 Cell Lines The following four CRISPR sgRNAs were used to target the first code exons of CD47 in HUES8 cells.
5'-gGTCCTGCCCTGTAACGGCGG-3'(SEQ ID NO: 75)
5'-gGACCGCCGCCGCGCGTCAC-3'(SEQ ID NO: 76)
5'-gCAGCAAGCGCCGCTCACC-3'(SEQ ID NO: 77)
5'-gTTCGCCCCCGCGGGGCGTGT-3'(SEQ ID NO: 78)

Cells were stained 72 hours after electroporation with anti-CD47 antibody (clone CC2C6) and CD47 negative cells were isolated using FACS Maria (BD). Colonies derived from single cells were obtained as previously described (Peters et al., 2013), followed by confirmation of deletion of CD47 expression by FACS analysis. Similarly, a beta-2-microglobulin (B2M) -deficient HUES8 cell line was generated using the following sgRNA:
5'-gCTACTCTCTTTTCTGCC-3'. (SEQ ID NO: 79)

Lentivirus transduction A doxycycline-inducible lentivirus gateway vector (Invitrogen) with an ORF of PD-L1 was constructed by PCR amplification. The PD-L1 expression lentivirus was packaged by transfecting HEK293T cells with the PD-L1 expression vector and the packaged plasmids pMDL, pVSVG, and pREV. Medium containing lentivirus particles was collected 48 hours after transduction and used to transduce VSMC with lentivirus particles encoding the doxycycline-binding transactivator rtTA. After 24 hours, VSMC was treated with doxycycline (10 μg / ml) to induce PD-L1 expression. This expression was verified by FACS. Evaluation of T cell proliferation for VSMC overexpressing PD-L1 was performed as described above, with the exception of 7-day co-incubation and the presence of doxycycline throughout the co-incubation. No effect of the addition of doxycycline on T cell proliferation was observed.

PBS containing 0.05% Immunofluorescence Tween®-20 was the wash buffer between each step after cell fixation. Briefly, cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were blocked overnight at 4 ° C. with 4% donkey serum (Jackson ImmunoResearch Laboratories) and incubated with the appropriate primary antibody diluted in block buffer for 1 hour at room temperature. The cells were then incubated with Alexa Fluor® 488 or Alexa Fluor® 555 binding secondary antibody (Life Technologies). The cells were washed and the nuclei were stained with Hoechst. The image was visualized with a Nikon inverted microscope.

RNA isolation, cDNA synthesis, and qPCR
RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the instruction manual. cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. QPCR using SYBR green or TaqMan was performed, relative quantities were specified using the QuantStudio 12k Flex System (Thermo Fisher Scientific), and then the relative Ct method (2 ^-ΔΔCt ) was used for each internal standard. Calculated as a relative value to.

Off-target analysis based on next-generation sequencing (NGS) Target external positions were predicted using CCTop (Stemmer et al., 2015). Bait design, genomic DNA enrichment (library preparation), and NGS were performed by Arbor Biosciences using the myBaits® custom target capture kit. Briefly, for each of the 648 predicted target external positions, 5 RNA baits were designed across each target external position and placed approximately every 26 bp to cover the range of 181 to 182 bp. After genomic DNA was extracted from the WT and the three modified hPSC strains, the biotinylated RNA bait was hybridized with the corresponding denatured genomic DNA library. Subsequently, the RNA-gDNA hybrid was bound to streptavidin-coated beads to wash off non-specific binding. The remaining gDNA library was amplified and sequenced by pair-end NGS using NovaSeq (Illumina).

Genome editing events were quantified by CRISPRessoPoled of the CRISPResso set (Version 1.0.13), which was used in the initial settings unless otherwise stated (Pinello et al., 2016). Briefly, for each of the four libraries, a read with a minimum single base pair score (phred33) greater than 25 was selected for a ± 100 bp width around each gRNA target external position of the human genome (hg38). Aligned. Regardless of the library, sites (= 3) with less than 5 aligned reads were excluded. The percentage of reads with modified sequences (insertions, deletions, and substitutions) relative to hg38 at each target external position from each library was calculated programmatically. If the% of reads with the modified sequence was found to be> 0 in WT and all three modified strains, the sequence was further investigated. When the sequences of all three modified strains and the WT sequence matched, the sequence was classified as SNP / PM. However, if the sequence of the modified cell line deviates from the WT sequence, the sequence was identified as an editing event. Polymorphism (PM) represents a small deletion / insertion in place of the single nucleotide polymorphism (SNP) already observed in WT's hPSC, which deviates from hg38.

Statistical analysis Plots were created and statistical analysis was performed using Prism 7 (Graphpad).

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Claims (76)

MHC-I and MHC-II human leukocyte antigen expression is reduced compared to wild-type stem cells, and tolerance-causing factor expression is increased compared to wild-type stem cells.
The MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C.
The MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, and HLA-DR, and the tolerant factor is CD47.
The stem cells. The stem cell of claim 1, wherein the reduced expression of the MHC-I human leukocyte antigen comprises removing the MHC-I human leukocyte antigen from at least one allele of the cell. The stem cell of claim 1 or 2, wherein the reduced expression of the MHC-II human leukocyte antigen comprises the introduction of one or more indels into CIITA. The stem cell according to any one of claims 1 to 3, further comprising a decrease in expression of CIITA. The stem cell according to any one of claims 1 to 4, wherein the tolerant factor is inserted into the safe harbor locus of at least one allele of the cell. The stem cell according to any one of claims 1 to 5, wherein the stem cell does not express HLA-A, HLA-B, and HLA-C. The stem cell according to any one of claims 1 to 6, wherein the stem cell does not express HLA-DP, HLA-DQ, and HLA-DR. The stem cell according to any one of claims 1 to 7, wherein the stem cell does not express CIITA. The stem cell according to any one of claims 1 to 8, wherein the tolerant factor further comprises HLA-G and / or PD-L1. Stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR and express CD47. The stem cell according to claim 10, wherein the cells are CIITA indel / indel, HLA-A-/-, HLA-B-/-, and HLA-C-/-stem cells. A method for preparing hypoimmunogenic stem cells, which reduces the expression of MHC-I and MHC-II human leukocyte antigens in stem cells and increases the expression of tolerant factors in the stem cells, thereby the hypoimmunogen. The method described above comprising preparing sex stem cells.
The MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C.
The MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, and HLA-DR, and the tolerant factor is CD47.
The method. The method of claim 12, wherein reducing the expression of the MHC-I human leukocyte antigen comprises removing the MHC-I human leukocyte antigen from at least one allele of the stem cells. 12. The method of claim 12 or 13, wherein reducing the expression of the MHC-II human leukocyte antigen comprises introducing one or more indels into CIITA. The method of any one of claims 12-14, further comprising reducing the expression of CIITA. The expression of any one of claims 12 to 15, wherein increasing the expression of the tolerant factor comprises inserting the tolerant factor into the safe harbor locus of at least one allele of the stem cell. Method. The method of any one of claims 12-16, wherein the tolerant factor further comprises PD-L1 and / or HLA-G. The method according to any one of claims 12 to 17, wherein the hypoimmunogenic stem cells do not express HLA-A, HLA-B, and HLA-C. The method according to any one of claims 12 to 18, wherein the hypoimmunogenic stem cells do not express HLA-DP, HLA-DQ, and HLA-DR. The method according to any one of claims 12 to 19, wherein the hypoimmunogenic stem cells do not express CIITA. Stem cells in which the expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant factors is regulated compared to wild-type stem cells. 21. The stem cell of claim 21, wherein the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C. 23. The stem cell of claim 21 or 22, wherein the regulated expression of the one or more MHC-I human leukocyte antigens comprises a reduction in the expression of the one or more MHC-I human leukocyte antigens. 23. 23. The one or more MHC-I human leukocyte antigens are deleted from the genome of the cell, whereby the expression of the one or more MHC-I human leukocyte antigens is regulated. The stem cell according to any one item. The stem cell according to any one of claims 21 to 24, wherein the one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. The regulated expression of the one or more MHC-II human leukocyte antigens is any one of claims 21-25, comprising reduced expression of the one or more MHC-II human leukocyte antigens. Described stem cells. 23. One of claims 21-26, wherein the insertion of one or more indels into CIITA thereby regulates the expression of the one or more MHC-II human leukocyte antigens. Stem cells. The stem cell according to any one of claims 21 to 27, wherein the cell does not express HLA-A, HLA-B, and HLA-C. The stem cell according to any one of claims 21 to 28, wherein the cell is HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , and CIITA ^{indel / indel} cell. The stem cell according to any one of claims 21 to 29, wherein the one or more tolerant factors are selected from the group consisting of HLA-G, PD-L1 and CD47. The stem cell according to any one of claims 21 to 30, wherein the regulated expression of the one or more tolerant factors comprises increased expression of the one or more tolerant factors. The stem cell according to any one of claims 21 to 31, wherein the one or more tolerant factors are inserted into the AAVS1 safe harbor locus. The stem cell according to any one of claims 21 to 32, wherein HLA-G, PD-L1 and CD47 are inserted at the AAVS1 safe harbor locus. The stem cell according to any one of claims 21 to 33, wherein the one or more tolerant factors inhibit immune rejection. The stem cell according to any one of claims 21 to 34, wherein the stem cell is an embryonic stem cell. The stem cell according to any one of claims 21 to 34, wherein the stem cell is a pluripotent stem cell. The stem cell according to any one of claims 21 to 34, wherein the stem cell is hypoimmunogenic. The stem cell according to any one of claims 21 to 34, wherein the stem cell is a human stem cell. The stem cell according to any one of claims 21 to 38, wherein the stem cell retains pluripotency. The stem cell according to any one of claims 21 to 39, wherein the stem cell retains a differentiation potential. The stem cell according to any one of claims 21 to 40, wherein the stem cell exhibits a decrease in T cell response. The stem cell according to any one of claims 21 to 41, wherein the stem cell exhibits protection from an NK cell response. The stem cell according to any one of claims 21 to 42, wherein the stem cell exhibits a decrease in macrophage phagocytosis. Stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR. The stem cell according to claim 44, wherein the stem cell is HLA-A ^{− / −} , HLA − B ^{− / −} , HLA—C ^{− / −} , and CIITA ^{indel / indel} cell. The stem cell according to claim 44 or 45, wherein the stem cell expresses the tolerant factors HLA-G, PD-L1 and CD47. The stem cell of claim 46, wherein the tolerant factor is inserted at the AAVS1 safe harbor locus. The stem cell according to claim 46 or 47, wherein the tolerant factor inhibits immune rejection. The stem cell according to any one of claims 44 to 48, wherein the stem cell is an embryonic stem cell. The stem cell according to any one of claims 44 to 48, wherein the stem cell is a pluripotent stem cell. The stem cell according to any one of claims 44 to 48, wherein the stem cell is hypoimmunogenic. A method for preparing hypoimmunogenic stem cells, which regulates one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant factors of stem cells as compared to wild-type stem cells. The method comprising preparing the hypoimmunogenic stem cells. 52. The method of claim 52, wherein the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C. 52 or 53. The method of claim 52 or 53, wherein the regulated expression of the one or more MHC-I human leukocyte antigens comprises a reduction in the expression of the one or more MHC-I human leukocyte antigens. 52 to 54, wherein the one or more MHC-I human leukocyte antigens are deleted from the stem cell genome, thereby regulating the expression of the one or more MHC-I human leukocyte antigens. The method according to any one. The method according to any one of claims 52 to 55, wherein the one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. The regulated expression of the one or more MHC-II human leukocyte antigens is any one of claims 52-56, comprising reduced expression of the one or more MHC-II human leukocyte antigens. The method described. 15. One of claims 52-57, wherein the insertion of one or more indels into CIITA thereby regulates the expression of the one or more MHC-II human leukocyte antigens. Method. The method according to any one of claims 52 to 58, wherein the hypoimmunogenic stem cells do not express HLA-A, HLA-B, and HLA-C. ^{13 . _ _} the method of. The method according to any one of claims 52 to 60, wherein the one or more tolerant factors are selected from the group consisting of HLA-G, PD-L1 and CD47. The method of any one of claims 52-61, wherein the regulated expression of the one or more tolerant factors comprises increased expression of the one or more tolerant factors. The method of any one of claims 52-62, wherein the one or more tolerant factors are inserted at the AAVS1 safe harbor locus. The method according to any one of claims 52 to 63, wherein HLA-G, PD-L1 and CD47 are inserted at the AAVS1 safe harbor locus. The method according to any one of claims 52 to 64, wherein the one or more tolerant factors inhibit immune rejection. The method according to any one of claims 52 to 65, wherein the hypoimmunogenic stem cells retain pluripotency. The method according to any one of claims 52 to 66, wherein the hypoimmunogenic stem cells retain the ability to differentiate. The method according to any one of claims 52 to 67, wherein the hypoimmunogenic stem cells exhibit a reduced T cell response. The method according to any one of claims 52 to 68, wherein the hypoimmunogenic stem cells exhibit protection from an NK cell response. The method according to any one of claims 52 to 69, wherein the hypoimmunogenic stem cells exhibit reduced macrophage phagocytosis. The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 1-2 so that the HLA-A gene is edited and said. 52. The method of claim 52, which reduces or eliminates HLA-A surface expression and / or activity in stem cells. The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 3-4, whereby the HLA-B gene is edited and said. 52. The method of claim 52, which reduces or eliminates HLA-B surface expression and / or activity in stem cells. The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 5-6, whereby the HLA-C gene is edited and said. 52. The method of claim 52, which reduces or eliminates HLA-C surface expression and / or activity in stem cells. The stem cells are brought into contact with the Cas protein or the nucleic acid sequence encoding the Cas protein and the ribonucleic acid having the sequence of SEQ ID NO: 7, thereby introducing Indel into CIITA and MHC-II human leukocyte antigen in the stem cells. 52. The method of claim 52, wherein the surface expression and / or activity is reduced or eliminated. A method of preparing hypoimmunogenic stem cells, which regulates one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant factors of stem cells as compared to wild-type stem cells. Including preparing the hypoimmunogenic stem cells by
The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences of SEQ ID NOs: 1-2 so that the HLA-A gene is edited and said. Decreases or eliminates HLA-A surface expression and / or activity in stem cells,
The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acids having the sequences of SEQ ID NOs: 3-4, whereby the HLA-B gene is edited and said. Decreases or eliminates HLA-B surface expression and / or activity in stem cells,
The stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and a third pair of ribonucleic acids having the sequences of SEQ ID NOs: 5-6, thereby editing the HLA-C gene and said. HLA-C surface expression and / or activity in stem cells is reduced or eliminated, and the stem cells are contacted with the Cas protein or the nucleic acid sequence encoding the Cas protein and the ribonucleic acid having the sequence of SEQ ID NO: 7. Thereby, Indel is introduced into CIITA to reduce or eliminate MHC-II human leukocyte antigen surface expression and / or activity in said stem cells.
The method. A method of transplanting at least one hypoimmogenic stem cell into a patient, wherein the hypoimmunogenic stem cell is one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerant sources. The method described above, wherein the expression of sex factors is regulated compared to wild-type stem cells.

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