CN113906048A - Universal donor stem cells and related methods - Google Patents

Abstract

Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein can be used to overcome immune rejection in cell-based transplantation therapy. In certain embodiments, the universal donor stem cells disclosed herein have regulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors.

Description

Universal donor stem cells and related methods

Cross Reference to Related Applications

This application is a continuation of U.S. application No. 16/596,697 filed on 8/10/2019, U.S. application No. 16/596,697 is a continuation of U.S. application No. 16/277,913 filed on 15/2/2019, and U.S. application No. 16/277,913 claims the benefit of U.S. provisional application No. 62/631,393 filed on 15/2/2018. The entire teachings of the above application are incorporated herein by reference.

Background

Therapies using human pluripotent stem cell-derived cells for transplantation have the potential to drastically alter the therapeutic modalities of disease. The major obstacle to their clinical transformation is the rejection of allogeneic cells by the recipient immune system. Strategies aimed at overcoming this immune barrier include the pooling of cells with defined HLA haplotypes (Nakajima et al, 2007; Taylor et al, 2005) and the generation of patient-specific induced pluripotent stem cells (ipscs) (Takahashi et al, 2007; Yu et al, 2007). However, various limitations (de Rham and Villard, 2014; Tapia and Scholer,2016) have prevented the widespread use of these methods and have emphasized the need for "off-the-shelf cellular products that can be readily administered to any patient in need.

Disclosure of Invention

Disclosed herein are effective strategies to overcome immune rejection in cell-based transplantation therapies by generating universal donor stem cell lines.

Disclosed herein are stem cells comprising modulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors, relative to wild-type stem cells.

In some embodiments, the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B and HLA-C. In some aspects, the modulated expression of one or more MHC-I human leukocyte antigens comprises a decrease in expression of one or more MHC-I human leukocyte antigens. In some embodiments, one or more MHC-I human leukocyte antigens are deleted from the genome of the cell, thereby modulating expression of the one or more MHC-I human leukocyte antigens.

In some embodiments, the one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ and HLA-DR. In some aspects, the modulated expression of one or more MHC-II human leukocyte antigens comprises a decrease in expression of one or more MHC-II human leukocyte antigens. In some embodiments, one or more indels are introduced into CIITA, thereby modulating 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 aspects, the cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell.

In some embodiments, the one or more tolerogenic factors are selected from HLA-G, PD-L1 and CD 47. In certain aspects, the modulated expression of one or more tolerogenic factors comprises increased expression of one or more tolerogenic factors. In some embodiments, one or more tolerogenic factors are inserted into the AAVS1 safety harbor locus. In some aspects, HLA-G, PD-L1 and CD47 are inserted into the AAVS1 safety harbor locus. In some embodiments, the one or more tolerogenic factors inhibit immune rejection.

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

In some embodiments, the stem cell retains pluripotency. In some aspects, the stem cells retain differentiation potential. In some embodiments, the stem cell exhibits a decreased T cell response. In some aspects, the stem cells exhibit protection from NK cell responses. In some embodiments, the stem cell exhibits 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 cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell. In some aspects, the stem cells express the tolerogenic factors HLA-G, PD-L1 and CD 47. In some embodiments, the tolerogenic factors are inserted into the AAVS1 safe harbor locus. In certain aspects, the tolerogenic factors inhibit immune rejection.

In some embodiments, the stem cell is an embryonic stem cell. In some aspects, the stem cell is a pluripotent stem cell. In some embodiments, the stem cells are less immunogenic.

Disclosed herein are methods of making a stem cell with reduced immunogenicity, the method comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of the stem cell relative to a wild-type stem cell, thereby making the stem cell with reduced immunogenicity.

In some embodiments, the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B and HLA-C. In some aspects, the modulated expression of one or more MHC-I human leukocyte antigens comprises a decrease in expression of one or more MHC-I human leukocyte antigens. In some embodiments, one or more MHC-I human leukocyte antigens are deleted from the genome of the stem cell, thereby modulating expression of the one or more MHC-I human leukocyte antigens.

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

In some aspects, the poorly immunogenic stem cells do not express HLA-A, HLA-B and HLA-C. In some embodiments, the low immunogenic stem cells areThe cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell.

In some embodiments, the one or more tolerogenic factors are selected from HLA-G, PD-L1 and CD 47. In some aspects, the modulated expression of one or more tolerogenic factors comprises increased expression of one or more tolerogenic factors. In some embodiments, one or more tolerogenic factors are inserted into the AAVS1 safety harbor locus. In some aspects, HLA-G, PD-L1 and CD47 are inserted into the AAVS1 safety harbor locus. In some embodiments, the one or more tolerogenic factors inhibit immune rejection.

In some embodiments, the low immunogenic stem cells retain pluripotency. In some aspects, the poorly immunogenic stem cells retain differentiation potential. In some embodiments, the poorly immunogenic stem cells exhibit a decreased T cell response. In some aspects, the hypoimmunogenic stem cells exhibit protection from NK cell responses. In some embodiments, the poorly immunogenic stem cells exhibit reduced macrophage phagocytosis.

In some embodiments, the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having the sequences SEQ ID NOs 1-2, thereby editing the HLA-a gene to reduce or eliminate HLA-a surface expression and/or activity in the stem cell. In some aspects, a stem cell is contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having sequences SEQ ID NOs 3-4, thereby editing an HLA-B gene to reduce or eliminate HLA-B surface expression and/or activity in the stem cell. In some aspects, a stem cell is contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having sequences SEQ ID NOs 5-6, thereby editing an HLA-C gene to reduce or eliminate HLA-C surface expression and/or activity in the stem cell. In some aspects, a stem cell is contacted with a Cas protein or a nucleic acid sequence encoding a Cas protein and a ribonucleic acid having the sequence of SEQ ID No. 7, thereby introducing an indel into CIITA to reduce or eliminate MHC-II human leukocyte antigen surface expression and/or activity in the stem cell.

Also disclosed herein are methods of making a poorly immunogenic stem cell, comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of the stem cell relative to a wild-type stem cell, thereby making a poorly immunogenic stem cell, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having sequences SEQ ID NOs 1-2, thereby editing the HLA-a gene to reduce or eliminate HLA-a surface expression and/or activity in the stem cell, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acids having sequences SEQ ID NOs 3-4, thereby editing the HLA-B gene to reduce or eliminate HLA-B surface expression and/or activity in the stem cell, wherein the stem cell is contacted with the Cas protein or a nucleic acid sequence encoding the Cas protein and a third pair of ribonucleic acids having the sequences SEQ ID NO:5-6, thereby editing the HLA-C gene to reduce or eliminate HLA-C surface expression and/or activity in the stem cell, and wherein the stem cell is contacted with the Cas protein or a nucleic acid sequence encoding the Cas protein and a ribonucleic acid having the sequence SEQ ID NO:7, thereby introducing an indel into CIITA to reduce or eliminate MHC-II human leukocyte antigen surface expression and/or activity in the stem cell.

Also disclosed herein are methods of transplanting at least one hypoimmunogenic stem cell into a patient, wherein the hypoimmunogenic stem cell comprises modulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors, relative to a wild-type stem cell.

Also disclosed herein are stem cells. The stem cells may comprise reduced expression of MHC-I and MHC-II human leukocyte antigens relative to wild-type stem cells and increased expression of tolerogenic factors relative to wild-type stem cells, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR, and wherein the tolerogenic factors are CD 47.

In some embodiments, the decreased expression of MHC-I human leukocyte antigens comprises a deletion of MHC-I human leukocyte antigens from at least one allele of the cell. In some embodiments, the reduction in expression of MHC-II human leukocyte antigens comprises introducing one or more indels into CIITA. In some embodiments, the stem cell further comprises reduced expression of CIITA. In some embodiments, the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell.

In some embodiments, the stem cells do not express HLA-A, HLA-B and HLA-C. In some embodiments, the stem cells do not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the stem cell does not express CIITA. In some embodiments, the tolerogenic factors further comprise 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 and express CD 47. In some embodiments, the cells are CIITAindel/indel, HLA-A-/-, HLA-B-/-and HLA-C-/-stem cells.

Also disclosed herein are methods of making the stem cells with reduced immunogenicity. The method can include reducing expression of MHC-I and MHC-II human leukocyte antigens of the stem cells and increasing expression of tolerogenic factors of the stem cells, thereby producing the hypoimmunogenic stem cells, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR, and wherein the tolerogenic factors are CD 47.

In some embodiments, reducing expression of MHC-I human leukocyte antigens comprises deleting MHC-I human leukocyte antigens from at least one allele of a stem cell. In some embodiments, reducing expression of MHC-II human leukocyte antigens 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 the tolerogenic factors comprises inserting the tolerogenic factors into the safe harbor locus of at least one allele of the stem cell.

In some embodiments, the tolerogenic factors further comprise PD-L1 and/or HLA-G.

In some embodiments, the low immunogenic stem cells do not express HLA-A, HLA-B and HLA-C. In some embodiments, the low immunogenic stem cells do not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the low immunogenic stem cells do not express CIITA.

Drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

FIGS. 1A-1J show that genome editing abrogates polymorphic HLA-A/-B/-C and HLA class II expression and enables the expression of immune regulators from the AAVS1 safety harbor locus. Figure 1A provides a schematic of HLA-B and HLA-C CRISPR/Cas9 knockout strategies. Each scissors represents two sgrnas. Purple, red and green arrows indicate primers used for PCR screening. FIG. 1B provides a schematic of an HLA-A knockout strategy. Each scissors represents one sgRNA. Yellow arrows show primers used for PCR screening. FIG. 1C provides a FACS contour plot showing successful elimination of HLA-A/B/C in HUES 8. Wild Type (WT) or HLA-A/B/C knock-out (KO) cells were treated with IFN γ for 48 hours before staining with the indicated antibodies. Figure 1D shows the targeting strategy for the CIITA locus. Blue arrows indicate primers used for PCR and Sanger sequencing. FIG. 1E shows differentiated CD144⁺HLA-DR Mean Fluorescence Intensity (MFI) in WT and KO EC. FIG. 1F provides a schematic depicting the genotypes of WT, KO, KI-PHC and KIPC cell lines. Figure 1G shows a knock-in strategy for immunomodulatory molecules. Scissors represent sgrnas targeting the AAVS1 locus. Black and grey arrows indicate primers used for PCR screening. FIG. 1H shows PD-L1 and HLA-G expression in KI-PHC cells. FIG. 1I shows CD47 expression in KI-PHC cells. MFI relative to WT cells is shown on the right side of the histogram. FIG. 1J shows PD-L1 and CD47 expression in KI-PC cells.

FIGS. 2A-2E show that the KO and KI cell lines retain pluripotency and differentiation potential. FIG. 2A shows immunofluorescence indicating expression of pluripotency markers by WT, KO, KI-PHC, and KI-PC human pluripotent stem cells (hPSCs). Scale bar, 200 μm. FIG. 2B shows that qRT-PCR was performed to measure three line markers after differentiation of WT, KO, KI-PHC and KI-PC hPSC into the three germ layers specified. Relative quantification was normalized to each gene level in unmodified hpscs. FIG. 2C shows that the chromosomal G-banding (G-banding) in the KO, KI-PHC and KI-PC cell lines exhibited normal karyotypes after several successive rounds of genomic engineering. Figure 2D provides a table showing PCR-based analysis of exon off-target sites in engineered hPSC lines. Figure 2E shows target capture sequencing results showing reads of% off-target site sequence changes in WT and engineered hPSC lines. Black circles, SNP/Polymorphism (PM) sites; red circle, edited off-target; blue circle, CIITA as positive control at target site.

FIGS. 3A-3D show a reduction in T cell activity against KO and KI-PHC cell lines in vitro. FIG. 3A provides a scatter plot showing CD3 when co-cultured with WT, KO or KI EC for 5 days⁺(left panel, n ═ 8 donors), CD4⁺(middle panel, n ═ 6 donors) and CD8⁺Percentage of proliferating T cells in the T cell population (right panel, n ═ 6 donors). T cells cultured alone were used as negative controls; t cells activated with CD3/CD28 beads served as positive controls. Paired one-way ANOVA followed by Tukey multiple comparison test was performed. Data are mean ± standard error of mean (sem);^*p<0.05；^**p<0.01. FIG. 3B provides a scatter plot showing CD3 after five days of co-culture with WT, KO or KI EC⁺(left panel), CD4⁺(middle panel) and CD8⁺CD69 in T cell population (right panel)⁺(upper panel) and CD25⁺Percentage of cells (lower panel) (n-11 donors in all panels). The same negative and positive controls were used as in fig. 3A. Paired one-way anova was performed followed by Tukey multiple comparison test. Data are mean ± standard error of mean;^**p<0.01；^***p<0.001；^****p<0.0001. FIG. 3C provides the data obtained from a representative donor at WT, KO or KI EC with^CD3+Bar graph of IFN γ (left panel) and IL-10 (right panel) concentrations in the medium after T cell co-culture. Spontaneous release from T cells alone was used as a negative control. A general one-way anova followed by Tukey multiple comparison test was performed. Data are mean ± standard deviation;^**p<0.01；^***p<0.001. fig. 3D provides a bar graph representing the percent T cell cytotoxicity against WT, KO and KI EC (n ═ 6 donors). LDH release assays were performed and the percentage of T cell cytotoxicity from each donor was calculated. Paired one-way anova was performed followed by Tukey multiple comparison test. Data are mean ± standard error of mean;^*p<0.05；^**p<0.01。

FIGS. 4A-4E show a reduction in T cell responses against KO and KI cell lines in vivo. FIG. 4A provides a schematic depicting allogeneic CD8⁺Work flow of T cell pre-sensitization and in vivo T cell recall response assay. Figure 4B shows the percentage increase in teratoma volume at day 5 or day 7 after T cell injection compared to day 0. Genotype of teratoma: WT (n ═ 9), KO (n ═ 7), KI-PHC (n ═ 6), and KI-PC (n ═ 7). A general one-way anova followed by Tukey multiple comparison test was performed. Data are mean ± standard error of mean;^*p<0.05. figure 4C shows the percentage increase in teratoma volume at day 0 of T cell injection compared to day 2 before injection. Genotype of teratoma: WT (n ═ 9), KO (n ═ 7), KI-PHC (n ═ 6), and KI-PC (n ═ 7). Fig. 4D shows relative hCD8 (left panel) and IL-2 (right panel) mRNA expression in WT (n-8), KO (n-7), KI-PHC (n-6), and KI-PC (n-7) teratomas harvested on day 8 post T cell injection. The expression is normalized to RPLP 0. A general one-way anova followed by Tukey multiple comparison test was performed. Data are mean ± standard error of mean;^*p<0.05；^**p<0.01. FIG. 4E shows representative hematoxylin and eosin (H) for WT, KO, KI-PHC and KI-PC teratomas harvested on day 8 post T cell injection&E) And (6) dyeing. Black arrows indicate sites of T cell infiltration. Scale bar, 100 μm.

Fig. 5A-5D show that the KI cell line is protected from NK cell and macrophage responses. Fig. 5A provides a scatter plot of NK cell degranulation against WT, KO or KI-PHC VSMC (n-7 donors). For each donor, the percentage of degranulated NK cells was plotted as% CD56 expressing CD107a⁺A cell. NK cells cultured alone were used as negativesComparison; NK cells treated with PMA/ionomycin served as positive controls. Paired one-way anova was performed followed by Tukey multiple comparison test. Data are mean ± standard error of mean;^**p<0.01. figure 5B provides a bar graph representing the percentage of NK cytotoxicity against WT, KO and KI-PHC VSMC from one representative donor at the indicated effector/target (E/T) ratio (n-3 replicates). LDH release assays were performed and% NK cytotoxicity was calculated as the specific lysis of the NK cell-killed VSMC relative to maximal cell lysis. Unpaired one-way anova was performed followed by Tukey multiple comparison test. Data are mean ± standard deviation;^*p<0.05；^***p<0.001. fig. 5C provides a time-lapse plot of a macrophage phagocytosis assay (n ═ 5 monocyte donors). The designated genotype of the pHrodo-red labeled VSMCs, either pretreated with staurosporine (STS) (right panel) or not (left panel), were incubated with monocyte derived macrophages for 6 hours. Images were acquired every 20min using the celldicover 7 live cell imaging system. The total integrated fluorescence intensity of the pHrodored + phagosomes for each image was analyzed. Data are mean ± standard error of mean. Fig. 5D provides a scatter plot of macrophage phagocytosis assay at 4 hours co-incubation (n ═ 9 monocyte donors, three independent experiments). The experimental conditions were the same as in fig. 5C. Paired one-way anova was performed followed by Tukey multiple comparison test. Data are mean ± standard error of mean;^*p<0.05；^**p<0.01. VSMC ═ vascular smooth muscle cells; NK ═ natural killer cells.

FIGS. 6A-6H show that genome editing abrogates polymorphic HLA-A/-B/-C and HLA class II expression and enables the expression of immune regulators from the AAVS1 safety harbor locus. FIG. 6A shows PCR confirmation of HLA-B/-C knockouts using the primer pair shown in FIG. 1A. FIG. 6B shows PCR confirmation of HLA-A knockouts using the primer set shown in FIG. 1B. FIG. 6C shows PCR products using primers flanking the CIITA cleavage site. Fig. 6D shows Sanger sequencing showing that in the KO cell line, 1bp (shown in red) was inserted on one CIITA allele, while 12bp (shown as a dashed line) was deleted from the other allele. Figure 6E shows CD144 expression in differentiated WT and KO Endothelial Cells (ECs). FIG. 6F shows a workflow for generating KO and KI ES cell lines. FIG. 6G shows PCR confirmation of knock-in KI-PHC/KI-PC constructs using the primers shown in FIG. 1G.

FIGS. 7A-7H show that genome editing abrogates polymorphic HLA-A/-B/-C and HLA class II expression and enables the expression of immune regulators from the AAVS1 safety harbor locus. FIG. 7A shows CD47 expression in WT and KI-PC ES cells. The MFI relative to WT cells is given on the right side of the histogram. FIG. 7B shows HLA-A2 expression in WT, KI-PHC and KI-PC ES cells following IFN γ treatment, confirming the elimination of classical HLA class Ia molecules in the KI cell line. FIG. 7C shows CD144 expression in differentiated WT, KI-PHC and KI-PC EC. FIG. 7D shows HLA-DR Mean Fluorescence Intensity (MFI), which confirms elimination of HLA class II in differentiated KI-PHC and KI-PC EC. In CD144⁺HLA-DR expression was analyzed on cells. FIG. 7E shows CD140b expression in differentiated WT, KO, KI-PHC and KI-PC VSMC. FIG. 7F provides a contour plot (upper left) showing the expression of PD-L1 and HLA-G in differentiated WT and KI-PHC VSMC. CD47 expression in differentiated WT and KI-PHC VSMC (upper right panel). Contour plots of PD-L1 and CD47 expression in differentiated WT and KI-PC VSMCs are shown (bottom left). CD47 expression in differentiated WT and KI-PC VSMC (lower right panel). FIG. 7G shows WT, B2M differentiated after IFN- γ stimulation^-/-KO and KI-PHC VSMC. Grey, isotype; color, antibody. FIG. 7H shows WT, B2M differentiated with and without IFN- γ stimulation^-/-Relative HLA-E mRNA expression in KO and KI-PHC VSMC.

FIG. 8 provides sequencing chromatograms predicting exon off-target sites in genetically modified hPSC lines and parental WT cells.

Figure 9 shows a sequence check from NGS showing editing of off-target sites in engineered hPSC lines, as well as SNPs/polymorphic sites observed in engineered lines and WT cells.

FIGS. 10A-10D show a reduction in T cell activity against KO and KI-PHC cell lines. FIG. 10A shows the use in a T cell proliferation and activation assayThe gating strategy of (1). FIG. 10B provides T cell proliferation assays using WT, KO and KI-PHC EC as one representative donor of target cells. CD3⁺(upper panel), CD4⁺(middle panel) and CD8⁺(lower panel). T cells cultured alone were used as negative controls; t cells treated with CD3/CD28 beads served as positive controls. Figure 10C shows doxycycline-inducible PD-L1 expression in WT VSMC. Figure 10D provides co-culture with VSMC in the presence or absence of doxycycline-induced PD-L1 expression for 7 days^CD3+(left panel), CD4⁺(middle panel) and CD8⁺Scatter plots of the percentage of proliferating T cells in the T cell population (right panel) (n-4 donors). T cells with reduced CFSE signal were quantified as proliferating cells. T cells cultured alone served as negative controls; t cells activated with CD3/CD28 beads were used as positive controls. Carrying out paired two-tail t test; data are mean ± standard error of mean;^*p<0.05; ns, no significance.

FIGS. 11A-11E show that the KI cell line is protected from the NK cell and macrophage responses. FIG. 11A shows a representative CD8⁺Expression of CD69 and PD-1 examined before and after priming (priming) of T donors. Figure 11B provides a gating strategy for NK cell degranulation assay. Figure 11C provides FACS contour plots for NK cell degranulation assays for one representative donor. FIG. 11D shows CD47 MFI, which confirms differentiated CD47^-/-Elimination of CD47 expression in VSMC. Figure 11E provides a fluorescence image showing phagocytic VSMCs from one representative donor pre-labeled with phododo-Red after 4h of incubation with macrophages. VSMC were pretreated with staurosporine, or not. Images represent the superposition of bright field and red channel, and the fluorescent phagosomes are highlighted after masking by ZEN imaging analysis software. Scale bar, 200 μm.

Fig. 12A-12C illustrate the overcoming of HLA barriers. Figure 12A provides a schematic of MHC class II and class I enhancers (enhanceosomes). Targeting CIITA (the primary regulator of MHC class II expression) prevents MHC class II expression. The promoter of the MHC class I gene is more complex, so deletion of NLRC5 (CIITA homolog regulating MHC class I expression) only results in a reduction of MHC class I expression. FIG. 12B shows IFNg-induced reduction of MHC class I expression in NLRC 5-/-CIITA-/hPSC. WT, or the designated KO hue 9 cells, were stimulated with IFNg for 48 hours and then stained for MHC class I expression, recorded by FACS. Deletion of the helper chain B2M completely prevented MHC class I surface expression, but made these cells susceptible to NK cell killing. FIG. 12C shows a targeting strategy for selective removal of polymorphic HLA genes HLA-A/B/C from the genome of hPSCs. A schematic of the targeting strategy is provided. PCR confirmation of each deletion in the genome of HUES8 is also shown.

FIGS. 13A-13E show the knock-in (KI) strategy for the tolerogenic factors into the safe harbor locus. FIGS. 13A-13B provide schematic diagrams of the KI construct. Fig. 13C shows confirmation of loss of HLA class I expression in two KI clones (C8 and C12). FIG. 13D shows the successful overexpression of PD-L1 and CD47 in HLA-deficient KI clones C8 and C12 from the AAVS1 safety harbor locus. Fig. 13E shows that the final goal is to reverse engineer the immunomodulatory activity of human trophoblasts (PD-L1, HLA-G, CD47 high) that induce tolerance to a semi-allogeneic fetus (50% derived from the father and therefore exogenous) during pregnancy.

FIGS. 14A-14B show functional immunosilent cells for transplantation. Figure 14A shows confirmation of HLA expression in modified hpscs (HUES 8). Loss of MHC class I expression was confirmed by FACS in two independent HLA-A/B/C-/-CIITA-/-KO clones, D1 and F2. Similar morphology of KO clone-derived Endothelial Cells (ECs) was observed. IFN γ -induced MHC class II expression in EC of the indicated genotype demonstrated loss of HLA class II in HLA-A/B/C-/-CIITA-/-KO clones. Figure 14B shows T cell proliferation assay (upper panel) and NK cell degranulation assay (lower panel). For the T cell proliferation assay (upper panel), CFSE labeled T cell clones were used to assess T cell proliferation against ECs derived from HUES9 of the indicated genotype. Loss of CFSE signal is proportional to T cell proliferation, which is a proxy for immune stimulatory activity of these cells. Although WT EC triggered significant T cell proliferation within 7 days, T cell proliferation decreased in the presence of two independent NLRC5-/-CIITA-/-KO clones and did not exist when co-incubated with B2M-/-CIITA-/-KO EC. For NK cell degranulation assay (lower panel), HLA-deficient VSMC (D1, F2) triggered enhanced NK cell degranulation when compared to WT cells. For NK degranulation, PMA/ionomycin or HLA-deficient 221 cells were used as positive controls. NC ═ negative control, NK cells only.

Fig. 15A-15B illustrate the generation of preclinical data. Figure 15A shows improved engraftment of immunosilent human pluripotent stem cells in humanized mice. NSG mice reconstituted with the human immune system (BLT) are transplanted with ES cells of a given genotype and allowed to form teratomas. Teratoma size and consistency at 4-6 weeks post-transplantation were scored blindly. Although WT teratomas showed signs of rejection, NLCR 5-/-CIITA-/-and B2M-/-CIITA-/-stem cells were found to be less restricted in growth, indicating that they are immunoprotected. Figure 15B shows that the introduction of an inducible Caspase9(iCasp9) killing switch can eliminate cells after treatment with CID dimerization factor. iCasp9 kills the cartoon of the switch (left). Dose titration and time course of CID dimerization factor in transiently transfected 293T cells (right). Finally, the iCasp9 killing switch will be integrated into the safe harbor locus of the modified immune-silenced stem cell.

Detailed Description

The inventions disclosed herein employ genome editing techniques (e.g., CRISPR/Cas or TALEN systems) to reduce or eliminate expression of key immune genes or, in some cases, to insert tolerance-inducing factors into stem cells, rendering them and differentiated cells prepared therefrom less immunogenic and less susceptible to immune rejection by a subject in which such cells are transplanted.

As used herein to characterize cells, the term "low immunogenicity" generally means that such cells are not susceptible to immunological rejection by a subject in which such cells are transplanted. For example, the propensity of such low immunogenic cells to be immunorejected by a subject transplanted with such cells may be reduced by about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more relative to unaltered wild type cells. In some aspects, genome editing techniques (e.g., CRISPR/Cas or TALEN systems) are used to modulate (e.g., reduce or eliminate) expression of MHC-I and MHC-II genes.

In certain embodiments, the invention disclosed herein relates to stem cells whose genome has been altered to reduce or delete key components of HLA expression. Similarly, in certain embodiments, the invention disclosed herein relates to stem cells whose genome has been altered to insert one or more tolerance-inducing factors. The present invention contemplates altering the target polynucleotide sequence in any manner available to those skilled in the art, for example, using TALENs, ZFNs, or CRISPR/Cas systems. Such CRISPR/Cas systems can employ a variety of Cas proteins (Haft et al PLoS Compout biol.2005; 1(6) e 60). 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. It should be understood that although examples of methods utilizing CRISPR/Cas (e.g., Cas9 and Cpf1) and TALENs are described in detail herein, the invention is not limited to the use of these methods/systems. Other methods of targeting polynucleotide sequences known to those skilled in the art to reduce or eliminate expression in a target cell may be utilized herein.

The present invention contemplates altering, e.g., modifying or cleaving, a target polynucleotide sequence in a cell for any purpose, but in particular such that expression or activity of the encoded product is reduced or eliminated. In some embodiments, the target polynucleotide sequence in a cell (e.g., an ES cell or iPSC) is altered to produce a mutant cell. As used herein, "mutant cell" generally refers to a cell that has a resulting genotype that is different from its original genotype or wild-type cell. In some cases, for example when a CRISPR/Cas system is used to alter a normally functioning stem gene, a "mutant cell" exhibits a mutant phenotype. In some embodiments, the target polynucleotide sequence in the cell is altered to correct or repair the genetic mutation (e.g., restore the normal phenotype of the cell). In some embodiments, the target polynucleotide sequence in the cell is altered to induce a gene mutation (e.g., to disrupt the function of a gene or genomic element).

In some embodiments, the alteration is an indel. As used herein, "indel" refers to a mutation caused by an insertion, a deletion, or a combination thereof. One skilled in the art will appreciate that indels in the coding region of the genomic sequence will result in frame shift mutations unless the length of the indels is a multiple of three. In some embodiments, the alteration 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 a target polynucleotide sequence.

In some embodiments, the alteration results in a knockout of the target polynucleotide sequence or portion thereof. For example, target polynucleotide sequences in cells can be knocked out in vitro, in vivo, or ex vivo for therapeutic and research purposes. Knocking out a target polynucleotide sequence in a cell can be used to treat or prevent a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in the cell ex vivo and introducing those cells comprising the knocked-out mutant allele into a subject).

As used herein, "knockout" includes deletion of 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 alteration results in decreased expression of the target polynucleotide sequence. The terms "reduced", "decrease" and "reduction" are used generically herein to mean a reduction by a statistically significant amount. However, for the avoidance of doubt, "reduced", "lowering", "reduction" includes a reduction by at least 10% compared to a reference level, for example a reduction by 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 up to and including a reduction of 100% (i.e. a level not present compared to a reference sample), or any reduction between 10% and 100%.

The terms "increased", "increase" or "enhancement" or "activation" are used generically herein to mean an increase in a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhancement" or "activation" mean an increase of at least 10% compared to a reference level, for example 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% compared to a reference level, or up to and including 100% increase, or any increase between 10% and 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or greater compared to a reference level.

The term "statistically significant" or "significantly" refers to statistical significance, and generally denotes two standard deviations (2SD) of marker concentration below normal or lower. The term refers to the statistical argument that there are differences. It is defined as the probability of making a decision to reject a null hypothesis when the null hypothesis is actually true. The decision is typically made using the p-value.

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

In some embodiments, the alteration results in correcting the target polynucleotide sequence from an undesired sequence to a desired sequence. The CRISPR/Cas system can be used to correct any type of mutation or error in a target polynucleotide sequence. For example, the CRISPR/Cas system can be used to insert nucleotide sequences that are missing from a target polynucleotide sequence due to a deletion. Due to insertion mutations, the CRISPR/Cas system can also be used to delete or excise a nucleotide sequence from a target polynucleotide sequence. In some cases, the CRISPR/Cas system can be used to replace an incorrect nucleotide sequence with the correct nucleotide sequence (e.g., to restore the function of a target polynucleotide sequence that is impaired due to a loss-of-function mutation).

The CRISPR/Cas system can alter target polynucleotides with surprisingly high efficiency. In certain embodiments, the altered efficiency is at least about 5%. In certain embodiments, the altered efficiency is at least about 10%. In certain embodiments, the altered efficiency is from about 10% to about 80%. In certain embodiments, the altered efficiency is from about 30% to about 80%. In certain embodiments, the altered efficiency is from about 50% to about 80%. In some embodiments, the altered efficiency is greater than or equal to about 80%. In some embodiments, the altered efficiency is greater than or equal to about 85%. In some embodiments, the altered efficiency is greater than or equal to about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the altered 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 genomic 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 comprises a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one to two ribonucleic acids (e.g., grnas) capable of directing and hybridizing the Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, the CRISPR/Cas system comprises a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one pair of ribonucleic acids (e.g., grnas) capable of directing and hybridizing the Cas protein to a target motif of a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) joined by peptide bonds, and include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

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 comprise conservative amino acid substitutions. In some cases, the substitution and/or modification may prevent or reduce proteolytic degradation of the polypeptide in the cell and/or extend the half-life of the polypeptide. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise a naturally occurring amino acid. In some embodiments, the Cas protein may comprise alternative amino acids (e.g., D-amino acids, β -amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include certain moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas 9. In some embodiments, the Cas protein comprises a Cas protein of an escherichia coli subtype (also referred to as CASS 2). Exemplary Cas proteins of the e.coli subtype include, but are not limited to Cse1, Cse2, Cse3, Cse4, and Cas5 e. In some embodiments, the Cas protein comprises a Ypest subtype of Cas protein (also referred to as CASS 3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to, Csy1, Csy2, Csy3, and Csy 4. In some embodiments, the Cas protein comprises a Nmeni subtype of Cas protein (also referred to as CASS 4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to, Csn1 and Csn 2. In some embodiments, the Cas protein comprises a Dvulg subtype of Cas protein (also referred to as CASS 1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2 and Cas5 d. In some embodiments, the Cas protein comprises a tnepap subtype of Cas protein (also referred to as CASS 7). Exemplary Cas proteins of tnepap subtype include, but are not limited to Cst1, Cst2, Cas5 t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5 h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also referred to as CASS 5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5 a. In some embodiments, the Cas protein comprises a Mtube subtype Cas protein (also referred to as CASS 6). Exemplary Cas proteins of Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm 5. In some embodiments, the Cas protein comprises a RAMP modular Cas protein. Exemplary RAMP modular Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr 6.

In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is Cas9 or a functional portion thereof from any bacterial species. The Cas9 protein is a member of a type II CRISPR system, which typically includes a small transcoding rna (tracrrna), an endogenous ribonuclease 3(rnc), and a Cas protein. The Cas9 protein (also known as CRISPR-associated endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids. Cas9 contains 2 endonuclease domains, including a RuvC-like domain (residues 7-22, 759-.

In some embodiments, the Cas protein is a Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 or a functional portion thereof from any bacterial species. The Cpf1 protein is a member of the type V CRISPR system. The Cpf1 protein is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Cpf1 cleaves target DNA in a staggered pattern using single ribonuclease domains. Staggered DNA double strand breaks result in 4 or 5-nt 5' overhangs.

As used herein, a "functional portion" refers to a portion of a peptide that retains its ability to complex with at least one ribonucleic acid (e.g., a guide rna (grna)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked functional domains of a Cas9 protein selected from a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cpf1 protein functional domains selected from 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.

It is to be understood that the present invention contemplates various ways of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas 9). In some embodiments, the exogenous Cas protein may be introduced into the cell as a polypeptide. In certain embodiments, the Cas protein may be conjugated or fused to a cell penetrating polypeptide or peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates the uptake of a molecule into a cell. The cell penetrating polypeptide may contain a detectable label.

In certain embodiments, the Cas protein may be conjugated or fused to a charged protein (e.g., with a positive, negative, or overall neutral charge). Such linkage may be covalent. In some embodiments, the Cas protein may be fused to an ultra-positively charged GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al ACS Chem biol.2010; 5(8): 747-52). In certain embodiments, the Cas protein may be fused to a Protein Transduction Domain (PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas protein comprises a Cas polypeptide fused to a cell penetrating peptide. In some embodiments, the Cas protein comprises a Cas polypeptide fused to a PTD.

In some embodiments, the Cas protein may be introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (e.g., Cas9 or Cpf 1). The process of introducing the nucleic acid into the 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 a modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA (e.g., a synthetic, modified mRNA) as described herein.

In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding the at least one to two ribonucleic acids are introduced into the cell by viral transduction (e.g., lentiviral transduction).

In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic, modified mRNA) as described herein.

The methods of the invention contemplate the use of any ribonucleic acid capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises CRISPR RNA (crRNA). In some embodiments, the single ribonucleic acid comprises a guide RNA that directs and hybridizes to a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs and hybridizes to a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, one to both ribonucleic acids comprise a guide RNA that directs and hybridizes to a Cas protein to a target motif of a target polynucleotide sequence in a cell. As will be understood by those skilled in the art, the ribonucleic acids of the invention can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system and the sequence of the target polynucleotide employed. One to two ribonucleic acids may also be selected to minimize hybridization to nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, one to two ribonucleic acids hybridize to target motifs that contain at least two mismatches when compared to all other genomic nucleotide sequences in a cell. In some embodiments, one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in a cell. In some embodiments, one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein that flanks the mutant allele between the target motifs.

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

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

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

In some embodiments, the target motif is a DNA sequence of 17 to 23 nucleotides. 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 to two ribonucleic acids hybridize to target motifs that contain at least two mismatches when compared to all other genomic nucleotide sequences in a cell. In some embodiments, one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in a cell. One skilled in the art will appreciate that a variety of techniques can be used to select suitable target motifs for minimizing off-target effects (e.g., bioinformatic analysis). In some embodiments, one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein that flanks the mutant allele between the target motifs.

In some aspects, a gene editing system (e.g., TALEN, ZFN, CRISPR/Cas, etc.) is used to alter a target polynucleotide sequence in a cell to reduce or eliminate the expression and/or activity of one or more key immune genes in the cell. In some embodiments, the disclosure provides altering a target polynucleotide sequence in a cell to delete a contiguous stretch of genomic DNA (e.g., delete one or more key immune genes) from one or both alleles of the cell (e.g., using a CRISPR/Cas system). In some embodiments, the target polynucleotide sequence in the cell is altered to insert a genetic mutation in one or both alleles of the cell (e.g., using a CRISPR/Cas system). In other embodiments, the universal stem cells disclosed herein can be subjected to complementary genome editing methods (e.g., using the CRISPR/Cas system), whereby such stem cells are modified to delete contiguous segments of genomic DNA (e.g., key immune genes) from one or both alleles of the cell, and one or more tolerance-inducing factors (such as HLA-G, CD47 and/or PD-L1) are inserted into one or both alleles of the cell to locally suppress the immune system and improve graft implantation.

The universal stem cells disclosed herein can be used, for example, to diagnose, monitor, treat, and/or cure the presence or progression of a disease or disorder (e.g., type 1 diabetes or multiple sclerosis) in a subject. As used herein, "subject" means a human or an animal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a juvenile. In certain embodiments, the subject is treated in vivo, in vitro, and/or in utero. In certain aspects, a subject in need of treatment according to the methods disclosed herein has a disorder or is suspected of having such a disorder or has an increased risk of developing such a disorder. In some aspects, the universal stem cell is transplanted into a subject.

Provided herein are novel cells, compositions and methods useful for addressing such HLA-based immune rejection of transplanted cells.

Elimination of MHC class I and MHC class II genes

In certain aspects, the invention disclosed herein relates to genomic modifications of one or more target polynucleotide sequences of the stem cell genome that modulate expression of MHC-I and/or MHC-II human leukocyte antigens. In some aspects, the gene editing system is used to modify one or more target polynucleotide sequences. In some aspects, the CRISPR/Cas system is used to delete and/or introduce indels into one or more target polynucleotide sequences.

Effective removal of HLA barrier can be achieved by direct targeting of polymorphic HLA alleles (HLA-a, -B, -C) and/or deletion of MHC enhancer components essential for HLA expression, such as CIITA.

In certain embodiments, HLA expression is interfered with. In some aspects, HLA expression is interfered with by targeting individual HLA (e.g., knocking out expression of HLA-A, HLA-B and/or HLA-C) and/or targeting transcriptional regulators of HLA expression (e.g., CIITA). In some aspects, multiple HLAs may be targeted simultaneously. For example, HLA-B and HLA-C are adjacent and can be targeted simultaneously. In some aspects, HLA-B and HLA-C, as well as the promoters of these two genes, can be knocked out using a CRISPR/Cas-deleted 95kb stem cell genome. In some aspects, HLA-a, as well as the promoter of this gene, is knocked out using the CRISPR/Cas-deleted 13kb stem cell genome.

In certain aspects, the stem cells disclosed herein do not express one or more human leukocyte antigens corresponding to MHC-I and/or MHC-II (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR), and are therefore characterized as being of low immunogenicity. For example, in certain aspects, a stem cell disclosed herein has been modified such that the stem cell or a differentiated stem cell prepared therefrom does not express or exhibits reduced expression of one or more of the following MHC-I molecules: HLA-A, HLA-B and HLA-C. In some aspects, one or more of HLA-A, HLA-B and HLA-C can be "knocked out" of the cell. Cells having a knockout of an HLA-A gene, HLA-B gene, and/or HLA-C gene can exhibit reduced or eliminated expression of each knockout gene. In some aspects, the stem cells disclosed herein have been modified such that the stem cells or differentiated stem cells prepared therefrom do not express or exhibit reduced expression of one or more of the following MHC-II molecules: HLA-DP, HLA-DQ and HLA-DR. In some aspects, one or more indels are inserted into a transcriptional regulator of HLA class II expression (e.g., CIITA). Cells with indels inserted into CIITAs (e.g., targeting exon 1) may exhibit reduced or eliminated expression of HLA-DP, HLA-DQ and/or HLA-DR.

In some aspects, the disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which an HLA-a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. A contiguous segment of genomic DNA can be deleted by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from SEQ ID NOs 1-2.

In certain aspects, the disclosure provides a method for altering a target HLA-a sequence in a cell comprising contacting the HLA-a sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif of the target HLA-a polynucleotide sequence, wherein the target HLA-a polynucleotide sequence is cleaved, and wherein the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from SEQ ID NOs 1-2.

In some aspects, the disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which an HLA-B gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. A contiguous segment of genomic DNA can be deleted by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from SEQ ID NOs 3-4.

In certain aspects, the disclosure provides a method for altering a target HLA-B sequence in a cell comprising contacting the HLA-B sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize to a target motif of the target HLA-B polynucleotide sequence, wherein the target HLA-B polynucleotide sequence is cleaved, and wherein the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from SEQ ID NOs 3-4.

In some aspects, the disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which an HLA-C gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. A contiguous segment of genomic DNA can be deleted by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from SEQ ID NOs 5-6.

In certain aspects, the disclosure provides a method for altering a target HLA-C sequence in a cell comprising contacting the HLA-C sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif of the target HLA-C polynucleotide sequence, wherein the target HLA-C polynucleotide sequence is cleaved, and wherein the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from SEQ ID NOs 5-6.

In certain aspects, the disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which a class II transactivator (CIITA) gene has been edited to introduce one or more indels into exon 1, thereby reducing or eliminating surface expression of MHC class II molecules (e.g., HLA-DP, HLA-DQ and HLA-DR) in the cells or populations thereof. One or more indels can be introduced by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid consisting of SEQ ID NO: 7. In some aspects, exon 1 of CIITA is targeted by a ribonucleic acid consisting of SEQ ID NO. 7 and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from SEQ ID NO. 1-2.

In certain aspects, the disclosure provides a method for introducing one or more indels in a cell, comprising contacting a CIITA sequence (e.g., exon 1 of CIITA) with a Cas protein or a nucleic acid encoding the Cas protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif of a target CIITA polynucleotide sequence, wherein the one or more indels are introduced into exon 1 of the CIITA polynucleotide sequence, and wherein the ribonucleic acid has the sequence of SEQ ID NO: 7. In some aspects, exon 1 of CIITA is targeted by a ribonucleic acid consisting of SEQ ID NO. 7 and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from SEQ ID NO. 1-2.

Insertion of tolerogenic factors

In certain embodiments, one or more tolerogenic factors may be inserted or reinserted into a genome-edited stem cell line to generate immune-privileged (immune-privileged) universal donor stem cells. In certain embodiments, the universal stem cells disclosed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of HLA-G, PD-L1 and CD 47. Expression of such tolerogenic factors may inhibit immune rejection.

The present inventors have used genome editing systems, such as CRISPR/Cas assisted Homology Directed Repair (HDR) systems, to facilitate the insertion of tolerogenic factors into safe harbor loci, such as the AAVS1 locus, to positively suppress immune rejection. In some aspects, the donor plasmid comprises an HLA-G expression cassette. In some aspects, the donor plasmid comprises a PD-L1 expression cassette. In some aspects, the donor plasmid comprises a CD47 expression cassette. In certain aspects, the donor plasmid comprises PD-L1, HLA-G, and CD47 expression cassettes. In certain aspects, the donor plasmid comprises a PD-L1 and a CD47 expression cassette. The donor plasmid comprising the expression cassette can target the AAVS1 locus of a stem cell (e.g., a low immunogenic stem cell). In certain aspects, the donor plasmid targets ribonucleic acids to the AAVS1 locus of a poorly immunogenic stem cell, wherein the ribonucleic acids have the sequence of SEQ ID No. 8.

In some aspects, the disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which the stem cell genome has been modified to express HLA-G. In some aspects, the present disclosure provides methods for modifying the genome of a stem cell to express HLA-G. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate HLA-G insertion into a stem cell line.

In some aspects, the present disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which the stem cell genome has been modified to express PD-L1. In some aspects, the present disclosure provides methods for altering the genome of a stem cell to express PD-L1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of PD-L1 into a stem cell line.

In some aspects, the present disclosure provides stem cells (e.g., low immunogenic stem cells) or populations thereof comprising a genome in which the stem cell genome has been modified to express CD-47. In some aspects, the present disclosure provides methods for altering the genome of an stem cell to express CD-47. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of CD-47 into a stem cell line.

In some aspects, the disclosure provides low immunogenic stem cells (e.g., stem cells modified to have eliminated expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR) or populations thereof comprising a genome in which the stem cell genome has been modified to express PD-L1, HLA-G and CD 47. In some aspects, the disclosure provides methods for modifying the genome of a stem cell to express PD-L1, HLA-G, and CD 47.

In some aspects, the disclosure provides low immunogenic stem cells (e.g., stem cells modified to have abolished expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR) or populations thereof comprising a genome in which the stem cell genome has been modified to express PD-L1 and CD 47. In some aspects, the present disclosure provides methods for modifying the genome of a stem cell to express PD-L1 and CD 47.

Universal stem cells

In certain aspects, the invention disclosed herein relates to universal stem cells. The universal stem cells may comprise reduced expression of one or more MHC-I and MHC-II human leukocyte antigens and increased or over-expressed expression of one or more tolerogenic factors. In certain aspects, the universal stem cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell that exhibits increased expression of HLA-G, PD-L1 and CD 47.

In some aspects, a stem cell (e.g., a universal stem cell) described herein exhibits one or more characteristics. For example, stem cells retain differentiation potential, exhibit reduced T cell responses, exhibit protection from NK cell responses, and exhibit reduced macrophage phagocytosis.

The universal stem cell can retain pluripotency, perform trilineage differentiation and retain a normal karyotype. For example, universal stem cells can retain expression of one or more of NANOG, OCT4, SSEA3, and TRA-1-60. In some aspects, the universal stem cell differentiates into three germ layers (e.g., ectoderm, mesoderm, and endoderm) and maintains expression of all lineage markers.

In some aspects, the universal stem cell exhibits a reduction in T cell-mediated adaptive immune response. For example, T cells (e.g., CD 4)⁺And CD8⁺T cells) exhibit reduced priming and activation for universal stem cells. Furthermore, T cells exhibit reduced cytokine secretion against universal stem cells. Reduced expression of HLA-I and HLA-II molecules can lead to CD4 targeting universal cells⁺And CD8⁺T cell priming decreases. In some aspects, expression of PD-L1 also inhibits CD8⁺Activation of T cells.

In some embodiments, the universal stem cell is protected from NK cell-mediated rejection. Due to HLA-G expression, universal stem cells can be protected against NK cell-mediated rejection. In some embodiments, the universal stem cell exhibits reduced macrophage phagocytosis. Overexpression of CD47 and/or expression of PD-L1 in universal cells can minimize or inhibit macrophage phagocytosis by universal cells.

Some definitions

Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

As used herein, the term "comprising" is used to refer to compositions, methods, kits and individual components thereof that are essential to the present invention, but is open to the inclusion of unspecified elements whether or not essential.

As used herein, the term "consisting essentially of … …" refers to those elements required for a given embodiment. The terminology allows for the presence of additional elements that do not materially affect one or more of the basic and novel or functional features of this embodiment of the invention.

The term "consisting of … …" refers to compositions, methods, kits, and respective components thereof as described herein, which do not include any elements not listed in this description of embodiments.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". The term "about" when used in conjunction with a percentage may mean ± 1%.

The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is also understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values given for a nucleic acid or polypeptide are approximations and are provided for purposes of description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". The abbreviation "e.g. (e.g.)" is derived from latin-exempli gratia and is used herein to represent non-limiting examples. The abbreviation "e.g." is therefore synonymous with the term "e.g. (for example)".

The entire teachings of PCT application PCT/US2016/031551, filed 5, 9, 2016, and incorporated herein by reference. All identified patents and other publications are expressly incorporated herein by reference for the purpose of description and disclosure, e.g., the methods described in such publications can be used in conjunction with the present disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. In this regard, no admission is made that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and should not constitute any admission as to the correctness of the dates or contents of these documents.

To the extent not already indicated, those of ordinary skill in the art will understand that any of the various embodiments described and illustrated herein can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

The following examples illustrate some embodiments and aspects of the invention. Those skilled in the relevant art will appreciate that various modifications, additions, substitutions and the like can be made without departing from the spirit or scope of the invention and that such modifications and changes are encompassed within the scope of the invention as defined in the following claims. The following examples do not limit the invention in any way.

Illustration of

Therapies using human pluripotent stem cell-derived cells for transplantation have the potential to drastically alter the therapeutic modalities of disease. The major obstacle to their clinical transformation is the rejection of allogeneic cells by the recipient immune system. Aiming at overcoming this exemptionStrategies for epidemic barriers include the pooling of cells with defined HLA haplotypes (Nakajima et al, 2007; Taylor et al, 2005) and the generation of patient-specific Induced Pluripotent Stem Cells (iPSCs) (Takahashi et al, 2007; Yu et al, 2007). However, various limitations (de Rham and Villard, 2014; Tapia and Scholer,2016) have prevented the widespread use of these methods and have emphasized the need for "off-the-shelf cellular products that can be readily administered to any patient in need. As a first step in the production of such universal stem cell products, elimination of HLA class I is required to prevent presentation of cellular peptides to cytotoxic CD8⁺T cells, since HLA class I molecules are expressed in almost all nucleated cells. In addition, elimination of HLA class II needs to be considered, as they are also highly polymorphic and may be present in certain hPSC-derived donor cell types, especially professional Antigen Presenting Cells (APC) and Endothelial Cells (EC) following IFN γ stimulation (Ting and Trowsdale, 2002). Recently, the performance of the CRISPR/Cas9 genome editing system provides a tool to interfere with HLA class I expression in hPSCs or hematopoietic cells by knocking out helper chain β -2-microglobulin (B2M) (Mandal et al 2014; Mattapally et al 2018; Meissner et al 2014; Riolobos et al 2013; Wang et al 2015) and to eliminate HLA class II expression by targeting its transcriptional major regulator CIITA (Chen et al 2015; Mattapally et al 2018). However, deletion of B2M also prevented surface expression of the non-polymorphic non-classical HLA class Ib molecules HLA-E and HLA-G which are required for maintaining NK cell tolerance (Ferreira et al, 2017; Lee et al, 1998B). In addition, it has been found that B2M-deficient cells are still rejected by allogeneic CD8+ T cells (Glas et al, 1992). Thus, the deletion of the HLA-A/-B/-C gene alone may represent a more advantageous strategy to protect donor cells from CD8⁺T cell mediated cytotoxicity without loss of HLA class Ib protective function.

Other approaches that have been explored to produce "off-the-shelf" cell products include the expression of co-inhibitory molecules and the co-stimulatory signals required to block complete T cell activation beyond HLA-T Cell Receptor (TCR) engagement. 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 humanized mouse models (Rong et al, 2014). However, this approach leaves the HLA barrier intact, which can lead to hyperacute rejection of implanted cells precipitated by pre-existing anti-HLA antibodies (Iniotaki-Theodoraki, 2001; Masson et al, 2007). In addition, CTLA-4Ig may also impair the homeostasis and function of regulatory T cells (Tregs), potentially compromising the establishment of operational 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 initiating adaptive immune responses in many cases, including chronic transplant rejection. The major problem associated with B2M depletion is that this strategy leaves donor cells susceptible to NK cell-mediated killing due to "self-depletion" (Raulet, 2006). Recently, Gornaluse et al expressed the B2M-HLA-E fusion construct in B2M deficient cells to overcome NK cell mediated lysis (Gornaluse et al, 2017). However, this approach does not address NK cells lacking NKG2A (an inhibitory receptor for HLA-E), whose reactivity may still be of concern (Braud et al, 1998 a; Pegram et al, 2011). Thus, the NK cell inhibitory ligand HLA-G, expressed at the maternal-fetal interface during pregnancy, which acts through a variety of inhibitory receptors (Ferreira et al, 2017; Pazmay et al, 1996), may be a better candidate to completely overcome NK cell responses. In addition, macrophages that cause rejection of transplanted cells may be controlled by the expression of CD47, CD47 being a "don't eat me" signal that prevents the cells from being phagocytosed by macrophages (Chhabra et al, 2016; Jaiswal et al, 2009; Majeti et al, 2009). However, this approach has not been explored to protect hpscs and their differentiated derivatives from macrophage phagocytosis. Furthermore, no convincing strategy to target both adaptive and innate immunity has been proposed.

Here, it is demonstrated that the CRISPR/Cas9 system can be used to selectively excise genes encoding polymorphic HLA class I members HLA-A/-B/-C from the genome of hPSCs. Furthermore, its multiplexing capability allows the use of a single guide RNA targeting CIITA while eliminating HLA class II gene expression. The resulting polymorphic HLA-deficient, "immunoopaque" cells were further modified to express the immunomodulatory factors PD-L1, HLA-G and CD47, which target immune surveillance by T cells, NK cells and macrophages, respectively, further attenuating allogenic responses in vitro and in vivo. Combining these and other genetic modifications can ultimately produce a universal "off-the-shelf" cell product suitable for transplantation into any patient.

Results

Genome editing abrogation of polymorphic HLA-A/-B/-C and HLA class II expression

Given the high homology of human MHC class I genes HLA-A, HLA-B and HLA-C, designing specific short guide rnas (sgrnas) targeting the coding region of each gene using the CRISPR/Cas9 genome editing system proved challenging. Therefore, the non-coding regions adjacent to these genes were targeted using a two-way multiplexing strategy to excise all three genes simultaneously from the genome of the hPSC line (HUES 8). In the HLA locus, HLA-B and HLA-C are adjacent, while HLA-A is closer to the telomere. To simultaneously knock out the adjacent HLA-B and HLA-C genes, two sgrnas were designed at each site upstream of HLA-B and downstream of HLA-C (fig. 1A). The predicted 95kb deletion also includes promoters for two genes, defined as the H3K27Ac positive region on the UCSC Genome Browser (UCSC Genome Browser). To knock out the entire HLA-A gene, one sgRNA was designed upstream of HLA-A and another sgRNA was designed downstream of HLA-A (FIG. 1B). According to the UCSC genome browser, the predicted 13kb deletion includes the HLA-A promoter. Both deletions were confirmed by PCR amplicons spanning the predicted Cas9 cleavage site (fig. 6A-6B). The elimination of HLA-A/-B/-C proteins in the final HLA knock-out clone (KO) was verified by flow cytometry (FIG. 1C).

Targeting the major regulator of HLA class II expression, CIITA, is a well-documented strategy to eliminate expression of three highly polymorphic HLA class II alleles HLADP/-DQ/-DR collectively (Krawczyk and Reith, 2006; Reith and Mach, 2001). Sgrnas targeting CIITA exon 1 with high cleavage efficiency were previously reported (fig. 1D) (Ding et al, 2013). The sgRNA is used in combination with a sgRNA targeting an HLA-a gene. A pair of PCR primers flanking the cleavage site in the first exon of CIITA was used to amplify the region spanning the cleavage site. The PCR amplicons were Sanger sequenced to identify biallelic frameshifts (fig. 6C-6D). To demonstrate that targeting CIITA results in loss of HLA class II expression, WT and KO hpscs were differentiated into Endothelial Cells (ECs) using a previously published protocol (Patsch et al, 2015). Notably, differentiated WT and KO ECs expressed equivalent levels of the EC marker CD144(VE-Cadherin), indicating that the differentiation efficiency of the resulting cells was not affected by genome editing (fig. 6E). Importantly, induction of HLA-DR expression following IFN γ stimulation was abolished in KO EC (fig. 1E). KO hPSC clones with HLA-A-/-HLA-B-/-HLAC-/-CIITAindel/indel genotypes were generated according to the workflow depicted in FIG. 6F. In summary, the results demonstrate that multiplexing CRISPR/Cas9 genome editing allows for combined and highly specific elimination of polymorphic HLA class I and class II gene expression in hpscs.

Knock-in of immunomodulatory factors into HLA knock-out cell lines

It is hypothesized that elimination of polymorphic HLA class Ia and class II molecules eliminates T cell-mediated adaptive immune rejection. However, HLA knockout cells may still be susceptible to innate immune cells (such as NK cells and macrophages) involved in allogenic reactions, which facilitates the exploration of the role of introducing immunomodulatory factors based on the following rationale: 1) although the non-polymorphic HLA-E gene will remain intact, its surface expression may be severely impaired by the removal of the polymorphic HLA class I gene, since the major peptide presented by HLA-E is a leader peptide from other class I molecules (Braud et al, 1998 b). Thus, the inability to express any HLA class I other than HLA-E makes the donor cells susceptible to NK cell-mediated lysis. In order to protect the engineered cells from NK cells, HLA-G was attempted to be introduced into HLA knock-out cells. 2) Macrophages are attracted by cytokines secreted at the site of implantation and are initiated by antibody binding to phagocytose foreign cells. CD47, which binds to signal-regulatory protein alpha (SIRPa) on the macrophage surface, thus acting as a "don't eat me" signal, has been well documented to increase significantly in certain types of tumors and help them escape macrophage phagocytosis (Betancur et al, 2017; Jaiswal et al, 2009; Willingham et al, 2012; Zhao et al, 2016). Therefore, the aim was to overexpress CD47 in HLA knock-out cells. 3) HLA-G presents classical peptides derived from intracellular proteins to T cells (Diehl et al, 1996), which may re-expose the cell line to CD8+ T cell immune surveillance. In addition, γ δ T cells can directly recognize antigens and initiate cytotoxic responses (vantourourout and Hayday, 2013). To counteract any residual T cell response, it was decided to knock-in T cell checkpoint inhibitor PD-L1, which can engage PD-1 receptors on activated T cells, directly inhibiting T cell activity (Riley, 2009). In addition, PD-L1 expression may also help protect transplanted cells from innate immune rejection by inhibiting PD-1+ NK cells (Beldi-Ferchiou et al, 2016; DellacChiesa et al, 2016) and PD-1+ macrophages (Gordon et al, 2017).

To avoid random integration and positional effects on transgene expression, attempts were made to knock-in immunomodulatory factors into the AAVS1 safety harbor locus (Sadelain et al, 2011). Two donor plasmids were designed, one containing PD-L1; HLA-G; CD47 expression cassette and another containing PD-L1; CD47 expression cassettes, both driven by the CAGGS promoter, flanked by arms homologous to the AAVS1 locus (fig. 1G). The donor plasmid was electroporated into HLA-A-/-HLAB-/-HLA-C-/-CIITAindel/indel clones along with sgRNA targeting the AAVS1 locus. Integration of the expression cassette in the AAVS1 locus was verified by PCR (fig. 6G). Two clones were isolated according to the workflow in fig. 6F and analyzed by flow cytometry; one, named KI-PHC, expressed PD-L1, HLA-G but did not significantly overexpress CD47 (fig. 1H-fig. 1I), and the second, named KI-PC, expressed PD-L1 and showed elevated levels of CD47 (fig. 1J and fig. 7A) compared to WT cells. Surface HLA-a2 levels were examined in two KI clones by flow cytometry and HLA class Ia elimination was confirmed (fig. 7B). KI-PHC and KI-PC hPSC differentiated into CD144+ EC (FIG. 7C), and no HLA-DR expression was observed by flow cytometry after IFN γ stimulation (FIG. 7D). Thus, immunomodulatory factors were successfully inserted into the AAVS1 safe harbor locus for HLA class Ia and class II null cells. Overall, three engineered hPSC lines were generated: KO, KI-PHC and KI-PC (FIG. 1F).

Next, attempts were made to confirm transgene expression as well as HLA-E expression in derivatives of the engineered hPSC lines. To this end, engineered hpscs were differentiated into Vascular Smooth Muscle Cells (VSMCs). WT, KO, KI-PHC and KI-PC VSMC expressed equivalent levels of the VSMC marker CD140b (PDGFRB), confirming similar differentiation efficiency (FIG. 7E). In KI-PHC VSMC, a moderately high expression of PD-L1 and HLA-G was observed for the subgroup compared to WT VSMC, as well as the main group showing significantly elevated PD-L1 and HLA-G levels (fig. 7F). However, no increase in CD47 expression was observed in KI-PHC VSMC (fig. 7F), which may be the result of incomplete expression from the targeting cassette, where all three gene products were linked by 2A peptide (fig. 1G). Similarly, a moderately high level of small subpopulations of PD-L1 and CD47 and a high level of major population were observed in KI-PC VSMC compared to WT VSMC (fig. 7F).

When WT VSMCs were stimulated with IFN γ, they greatly up-regulated HLA-E surface expression. In contrast, HLA-E protein levels on the cell surface were greatly reduced in KO VSMC (FIG. 7G), which was not due to impaired HLA-E gene expression in KO VSMC (FIG. 7H). Surprisingly, surface HLA-E expression of KI-PHC VSMC was not restored by HLA-G expression (FIG. 7G). However, HLA-G surface trafficking was not impaired in KI-PHC VSMC (fig. 7F), which provides further incentive to introduce such tolerogenic factors into engineered cell products to compensate for the reduction of HLA-E surface expression in HLA-a/-B/-C null backgrounds.

KO and KI cell lines retain pluripotency and differentiation potential

To assess whether engineered hPSC lines retain pluripotency, expression of NANOG, OCT4, SSEA3, SSEA4 and TRA-1-60 was assessed by immunofluorescence on KO, KI-PHC and KI-PC hpscs and found to be equivalent to unmodified hpscs (fig. 2A). In addition, KO, KI-PHC and KI-PC hPSC were differentiated into three germ layers. qRT-PCR was performed to examine the expression of ectodermal, mesodermal and endodermal markers and compared to three germ layers derived from unmodified hpscs. All of the lineage markers analyzed were found to be expressed in their respective germ layer cells (fig. 2B). In addition, KO, KI-PHC and KI-PC hPSC showed normal karyotypes (FIG. 2C). Thus, despite multiple rounds of genetic modification, these engineered hPSC lines remain pluripotent, undergo trilineage differentiation, and retain normal karyotype.

To analyze the potential off-target effect of sgrnas used in engineered hPSC lines, the first 21 in silico predicted exon off-target sites were PCR amplified from the engineered hPSC lines as well as from the parent WT hPSC. Sanger sequencing of the PCR products did not show any unwanted editing at these sites except for the pseudogene HLA-h (hfe), which showed a perfect match to sgrnas upstream of HLA-a used to delete HLA-a from the genome (fig. 2D and fig. 8). More broadly, all 648 predicted off-target sites of the 8 sgrnas used in this study were subjected to target capture sequencing. After enrichment by specially designed RNA decoys, for each predicted off-target site, the enriched DNA fragments are sequenced by Next Generation Sequencing (NGS). Sequence reads for each cell line were aligned and compared to hg38 genomic reference sequences, and the percentage of reads with altered sequences was calculated. Thus, HLA-h (hfe) was identified as off-target in all three cell lines, except for the identification of 12 naturally occurring SNPs/polymorphic sites. In addition, intron-off-target sites due to targeting of HLA-C were detected in TRAF3 in all three cell lines, and intron-off-target sites due to AAVS1 sgRNA were detected in CPNE5 in the KI-PC cell line (fig. 2E and fig. 9). Overall, the engineered hPSC lines retained pluripotency and their ability to differentiate into cells of all three germ layers, as well as into VSMCs and ECs, with similar differentiation efficiency as their WT counterparts, despite the detection of three off-target events.

Reduction of T cell response to KO and KI cell lines

Given that removal of polymorphic HLA class Ia expression is expected to abrogate T cell-mediated adaptive immune responses, next attempts were made to study T cell activity in co-culture with engineered cell lines. In addition to KO cells, KI-PHC cells were also used to address whether expression of the T cell checkpoint inhibitor PD-L1 would further inhibit T cell activity. Four independent in vitro T cell immunoassays were performed: t cell proliferation, activation, cytokine secretion and killing assays. Since HLA I expression is modest in hPSCs (de Almeida et al, 2013; Drukker et al, 2002), engineered and WT hPSCs differentiate into ECs that express HLA I and HLA II upon IFN γ stimulation prior to use in respective immunoassays; or to VSMCs, which express HLA I only.

For the T cell proliferation assay, WT, KO and KI-PHC ECs were pretreated with IFN γ for 48 hours and then co-cultured with CFSE labeled allogeneic CD3+ T cells for five days. T cells were then stained for CD3/4/8 and dilutions of CFSE signals were analyzed by flow cytometry as readouts for T cell proliferation in different T cell subsets (fig. 10A). FACS plots for one representative T cell donor are shown in figure 10B. As predicted, the percentage of total proliferating T cells (CD3+) decreased when incubated with KO EC (4.17% ± 0.89% SEM) or KI-PHC EC (3.87% ± 0.73% SEM) compared to WT EC (8.29% ± 1.23% SEM) (fig. 3A, left panel). CD4+ T cells followed a similar pattern, with WT EC (5.03% ± 0.89% SEM) inducing more CD4+ T cell proliferation than KO EC (3.58% ± 0.86% SEM) or KI-PHC EC (3.49% ± 0.83% SEM) (fig. 3A, middle panel). Furthermore, CD8+ cytotoxic T cells showed significantly reduced proliferation when co-cultured with KO EC (7.71% ± 1.89% SEM) or KI-PHC EC (5.95% ± 1.48% SEM) compared to WT EC (14.32% ± 2.39% SEM) (fig. 3A, right panels). Importantly, proliferation of CD8+ T cells was significantly reduced in the presence of KI-PHC EC when compared to KO EC co-culture (fig. 3A, right panel), indicating that PD-L1 overexpression in HLA null background inhibited CD8+ T cell activation even further. To further investigate the inhibitory effect of PD-L1 on the response of different T cell subsets, hPSC lines inducibly expressing PD-L1 were generated and differentiated into ECs prior to T cell proliferation assays. It was found that only CD8+ T cell proliferation was reduced in the presence of EC expressing PD-L1, while CD4+ T cell proliferation was not reduced when compared to WT EC, demonstrating the specific inhibitory effect of PD-L1 on CD8+ T cell subpopulations (fig. 10C-fig. 10D).

Expression of the T cell activation markers CD25 and CD69 was examined using the same co-culture of T cells with EC as the target cells (fig. 3B). When compared to T cells co-incubated with WT EC (6.43% + -0.71% SEM; 9.30% + -1.51% SEM), a reduction in the percentage of CD25+ and CD69+ T cells (CD3+) was found in co-culture with KI-PHC EC (4.91% + -0.74% SEM; 5.04% + -1.24% SEM) or KO EC (5.12% + -0.77% SEM; 5.40% + -1.29% SEM) (FIG. 3B). The same trend was observed in the CD4+ and CD8+ cell populations (fig. 3B). It was also found that a higher percentage of CD25+ cells was observed in the CD4+ cell population, and a higher percentage of CD69+ cells was observed in the CD8+ cell population when co-cultured with WT EC. However, no significant reduction in expression of activation markers for KI-PHC ECs was observed in T cells when compared to KO ECs.

Next, the levels of T cell effector cytokines IFN γ and IL-10 secreted into the medium during five days of T cell-EC co-culture were examined. When T cells were exposed to KO (3214 + -180.5 SD; 5.09 + -0.16 SD) or KI-PHC EC (2635 + -132.9 SD; 3.56 + -0.63 SD), the levels of both cytokines in the medium were lower, compared to the IFN γ and IL-10 levels (4747 + -556.1 SD; 54.56 + -17.22 SD) observed in the medium after exposure to WT EC, indicating that the cytokine secretion by T cells against the KO or KI-PHC cell lines was reduced (FIG. 3C).

To quantify T cell killing, Lactate Dehydrogenase (LDH) released from VSMC was measured as a surrogate indicator for T cell cytotoxicity. In this case, whereas VSMC express only HLA I, it is expected that only CD8+ T cells will be activated by HLA I-TCR engagement. CD8+ T cells were found to be least cytotoxic against KI-PHC VSMC (15.31% ± 4.52% SEM) when compared to KO VSMC (18.86% ± 4.34% SEM) and WT VSMC (37.65% ± 7.64% SEM) (fig. 3D). This observation indicates that PD-L1 further inhibited CD8+ T cell cytotoxicity in KI-PHC VSMC, which is consistent with the results of the CD8+ T cell proliferation assay. Overall, observations in T cell immunoassays demonstrate that CD4+ and CD8+ T cells against KO and KI-PHC cell lines are initiating to decrease due to the removal of HLA I and II molecules. Expression of PD-L1 in the KIPHC cell line further inhibited CD8+ T cell activation.

To assess T cell responses in vivo, WT and engineered hpscs were subcutaneously transplanted into immunodeficient mice and allowed to form teratomas over the course of 4-6 weeks. Presensitized allogeneic CD8+ T cells were then adoptively transferred via tail vein injection, and teratoma growth was monitored for an additional 8 days (fig. 4A). T cells for injection were activated after priming (CD69+) and had no signs of depletion (PD-1+) as measured by CD69 and PD-1 expression of CD8+ T cells before and after priming (fig. 11A). Consistent with the hypothesis that WT cells alone would be rejected, WT teratomas showed a slower increase in volume compared to KO teratomas 7 days after CD8+ T cell injection, not due to the slower growth rate of the WT teratomas themselves (fig. 4B-4C). These results indicate that KO teratomas are protected from T cell mediated rejection. Furthermore, although not significant, the mean volume of KI-PHC and KI-PC teratomas was also greater than that of WT teratomas 7 days after T cell infusion (fig. 4B). Furthermore, teratomas derived from both KO and KI cell lines showed reduced T cell infiltration as demonstrated by qPCR (fig. 4D) and histology (fig. 4E) for the human effector T cell markers CD8 and IL-2. Taken together, these observations suggest that removal of polymorphic HLA molecules from the cell surface of transplanted cells can effectively block T cell-mediated rejection in vivo, which matches in vitro observations.

The KI cell line is protected from the NK cell and macrophage responses

KO hPSC and its derivatives are expected to be susceptible to NK cell-mediated lysis due to the absence of HLA Ia molecules and impaired HLA-E surface expression, whereas KI-PHC cell lines should be protected from NK cell-mediated rejection due to HLA-G expression. To test the hypothesis, allogeneic NK cells were isolated from healthy donors and co-incubated with WT, KO or KI-PHC VSMC. Surface expression of CD56+ NK cells as a degranulation marker CD107a was analyzed by flow cytometry as a readout for NK cell activation (fig. 11B). Notably, NK cell degranulation in the presence of KO VSMC was not significantly higher than WT VSMC (10.16% ± 2.96% SEM) (fig. 5A), indicating a lack of NK cell activation signals on hPSC-derived VSMC. However, consistent with the hypothesis, the percentage of CD107a + degranulated NK cells in coculture with KI-PHC VSMC (5.43% ± 0.95% SEM) was found to be significantly lower than in the presence of KO VSMC (13.51% ± 2.51% SEM) (fig. 5A), suggesting that NK cell activity was indeed inhibited by HLA-G expression in KI-PHC VSMC. FACS plots for one representative donor are shown in figure 11C. LDH released from apoptotic VSMCs after co-incubation with NK cells was also examined to quantify NK cell cytotoxicity. Consistent with NK cell degranulation, a decrease in NK cell cytotoxicity was observed when NK cells were incubated with KI-PHC VMSC (fig. 5B).

Finally, macrophage activity was examined using a pH sensitive fluorescent dye (pHrodo-Red), which emits a signal after phagocytosis. It is hypothesized that overexpression of the ' don't eat me ' signal CD47 by macrophages in derivatives of the engineered hPSC cell line would reduce macrophage phagocytosis. Whereas no significant increase in CD47 expression was observed in KI-PHC VSMC (fig. 7F), VSMC differentiated from KI-PC cell line were used for these assays, which showed CD47 levels much higher than WT VSMC (fig. 7F). In addition, a CD47 knock-out (CD47-/-) cell line was generated as a positive control for macrophage phagocytosis and the loss of CD47 cell surface expression was verified by flow cytometry (fig. 11D). pHrodo-Red labeled VSMCs differentiated from WT, CD 47-/-and KI-PC cells were treated with staurosporine (STS) to induce apoptosis, or left untreated, and then incubated with isolated allogeneic macrophages from healthy donors. The appearance of red signal (an indicator of VSMC phagocytosis by macrophages) was monitored by live cell imaging and the fluorescence intensity quantified. Notably, KI-PC VSMC showed a significant decrease in macrophage phagocytosis when compared to CD 47-/-or WT VSMC, regardless of whether treated with STS or not (fig. 5C-5D and 11E). These data demonstrate that overexpression of CD47 does minimize macrophage phagocytosis of engineered hPSC-derived VSMC, but does not exclude the contribution of PD-L1 to inhibition of macrophage phagocytosis, as is expressed by KI-PC VSMC (fig. 7F).

Discussion of the related Art

In this study, the use of multiplexed CRISPR/Cas9 genome editing knockouts highly polymorphic HLA-a/-B/-C genes and successfully prevented expression of HLA class II genes by targeting the CIITA gene in hpscs. Furthermore, CRIPSR/Cas9 assists Homology Directed Repair (HDR) for the introduction of immunomodulatory factors PD-L1, HLA-G and CD47 into the AAVS1 locus. Engineered hPSC derivatives were found to cause significantly less immune activation and killing of T cells and NK cells and showed minimal phagocytosis by macrophages.

In the method for eliminating HLA class Ia expression, the polymorphic HLA class Ia genes HLA-A/-B/-C are specifically excised while leaving the genes B2M and the non-polymorphic HLA class Ib genes HLA-E, -F and-G intact. Although the resulting 95kb deletion contained not only the HLA-B/-C gene but also MIR6891 and four pseudogenes, no changes in growth rate or differentiation efficiency were observed in the KO or KI cell lines. Interestingly, for unknown reasons, HLA-E surface expression was not restored by expression of HLA-G in KI-PHC cells, which is inconsistent with previous reports that leader peptides from HLA-G are sufficient to promote HLA-E surface trafficking (Lee et al, 1998 a).

HLA knock-out (KO) hPSC lines were generated by genome editing using seven different sgrnas, KI-PHC and KI-PC hPSC were clones derived from the KO line and edited by an additional sgRNA targeting the AAVS1 locus. Among the 648 predicted off-target sites of the 8 sgrnas used, only one exon off-target event was observed in the transcribed pseudogene HLA-H due to one of the sgrnas used to delete HLA-a from the genome. If translated, the observed 2bp deletion found in both alleles will result in a frameshift mutation. Although mutations in HLA-H are associated with hereditary hemochromatosis, a rare disorder of iron storage (Feder et al, 1996), the observed HLA-H (HFE) mutations did not affect the growth rate or differentiation efficiency of the cell types tested in this study. Notably, it is possible to avoid such off-target events by involving a different sgRNA or by genotyping the HLA-H locus to select clones that do not contain this particular off-target mutation. Furthermore, targeting using the sgRNA/Cas9 ribonucleoprotein complex (RNP), which allows more transient editing than plasmid-based methods (Roth et al, 2018), should reduce the number of off-target events per cell line, and thus be applied in the future.

As expected, ablation of polymorphic HLA expression in hpscs and derivatives thereof (such as EC and VSMC) resulted in a reduction of T cell responses in vitro and in vivo. An interesting observation from T cell assays is that overexpression of the checkpoint inhibitor PD-L1 has a significant effect only on the proliferation and cytotoxicity of CD8+ T cells. There are several possible explanations for this: 1) the level of PD-L1 receptor PD-1 on CD8+ T cells was higher than on CD4+ T cells. 2) CD8+ T cells are the most sensitive cell type in the assay to exposure to target cells and therefore also express higher levels of the negative regulator PD-1. Notably, the two explanations are not mutually exclusive, as the intensity of T cell activation and PD-1 expression are linked by a negative feedback loop (Riley, 2009). Furthermore, it should be noted that PD-L1 alone had an effect on CD8+ T cell proliferation even in the absence of HLA, suggesting that PD-L1 may act as a tolerogenic factor even in the absence of productive HLA-TCR interactions. Furthermore, in T cell activation and cytokine secretion assays, background T cell activity was observed even in co-culture with KIPHC cell lines when compared to negative controls. This may be due to experimental settings that take into account that target cells may secrete factors that promote T cell activation, regardless of the presence of HLA.

While acute transplant rejection is primarily T cell mediated, the role of other immune cells (such as macrophages, NK cells, and B cells) must also be considered in terms of engraftment and long-term survival of the therapeutic cells. NK cell assays indicate that HLA-G expression can control NK cell activity. Furthermore, overexpression of CD47 effectively reduced macrophage phagocytosis. However, as recently reported, co-expressed PD-L1 in both KI cell lines also affected the activity of PD-1+ NK cells (Beldi-Ferchiou et al, 2016; Della Chiesa et al, 2016) and PD-1+ macrophages (Gordon et al, 2017), which may contribute to the observed phenotype. In terms of long-term implantation, among other things, antibody-dependent cellular cytotoxicity (ADCC) of NK cells and alloantibody-mediated complement activation, which are major drivers of chronic transplant rejection, must be considered (Baldwin et al, 2016; Djamali et al, 2014; Michaels et al, 2003). It is envisioned that the introduction of additional factors known to inhibit ADCC and complement activation, such as CD59(Meri et al, 1990), may result in a durable implantation. Finally, in vivo experiments will help elucidate the degree of protection that modified cells can have after transplantation. However, although various humanized mouse models exist, they are limited in reproducing the complete human immune response. Thus, there may be a need to develop improved in vivo models for testing cell transplantation and rejection (Brehm et al, 2014; Li et al, 2018; Melkus et al, 2006; Rongvaux et al, 2014).

Overcoming the immune barrier of transplantation would provide an exciting new way not only to overcome the allogeneic barrier, but also to have the potential to treat autoimmune diseases such as type 1 diabetes (T1D) and multiple sclerosis, where one specific cell type is attacked by the patient's autoimmune system and needs to be replaced. Thus, the generation of universal cells that can be safely transplanted into any human is expected to release the full potential of regenerative medicine.

Experimental procedures

CRISPR gRNA sequence

HLA-A upstream: 5'-GCCGCCTCCCACTTGCGCT-3' (SEQ ID NO:1)

HLA-A downstream: 5'-CACATGCAGCCCACGAGCCG-3' (SEQ ID NO:2)

HLA-B upstream _ 1: 5'-ATCCCTAAATATGGTGTCC-3' (SEQ ID NO:3)

HLA-B upstream _ 2: 5'-TCCCTAAATATGGTGTCCCT-3' (SEQ ID NO:4)

HLA-C downstream _ 1: 5'-GTGATCCGGGTATGGGCAGT-3' (SEQ ID NO:5)

HLA-C downstream _ 2: 5'-TGATCCGGGTATGGGCAGTG-3' (SEQ ID NO:6)

CIITA：5'-TCCATCTGGTCATAGAAG-3'(SEQ ID NO:7)

gRNA_AAVS1-T2：5'-GGGGCCACTAGGGACAGGAT-3'(SEQ ID NO:8)

PCR and qPCR probes/primers

PCR primers used in fig. 6:

Purple_F：5'-CACTCAGAGCAAAGGTCAGATG-3'(SEQ ID NO:9)

Purple_R：5'-AGACTTGAATCCATAAGCCCAA-3'(SEQ ID NO:10)

Red_F：5'-GACAAGTCTCGGAGATGGTTTT-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'-TTTGTTGTCAGCCAGACATAGG-3'(SEQ ID NO:14)

Yellow_F：5'-CTGGTTATCTCCCCATTCTCTG-3'(SEQ ID NO:15)

Yellow_R：5'-AAGCATTCACTCCTGACCCTG-3'(SEQ ID NO:16)

Blue_F：5'-GTCTTCCCTCCCAGGCAGCTCA-3'(SEQ ID NO:17)

Blue_R：5'-TGAGGGGTGGGGGATACCGGA-3'(SEQ ID NO:18)

Black_F：5'-TCGACCTACTCTCTTCCGCA-3'(SEQ ID NO:19)

Black_R：5'-TAGGGGGCGTACTTGGCATA-3'(SEQ ID NO:20)

Gray_F：5'-CCGTTCTCCTGTGGATTCGG-3'(SEQ ID NO:21)

Gray_R：5'-TCTCTGGCTCCATCTAAGC-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'-CCACGGGCCAACTACTCC-3'(SEQ ID NO:26)

HEATR1_F：5'-TTACCCAGTTCAATACTGAGCCA-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'-CCGCTAGACCTGCTGCTATG-3'(SEQ ID NO:32)

ACSBG1_F：5'-CTGGGTGTCAATGATGGCGT-3'(SEQ ID NO:33)

ACSBG1_R：5'-GCCACATCTAAAGGCAGTCG-3'(SEQ ID NO:34)

AC078852.1_F：5'-GTTTGTGGGTGCTGTCAAC-3'(SEQ ID NO:35)

AC078852.1_R：5'-CTAGGCAACAGTGACAGGGG-3'(SEQ ID NO:36)

HIPK4_F：5'-GGACCATCATGTCGGAGACC-3'(SEQ ID NO:37)

HIPK4_R：5'-GACCTGGGGAGTCACACGAAC-3'(SEQ ID NO:38)

ACSBG1_F：5'-CTGGGTGTCAATGATGGCGT-3'(SEQ ID NO:39)

ACSBG1_R：5'-GCCACATCTAAAGGCAGTCG-3'(SEQ ID NO:40)

HIC2_F：5'-AAGTGTTCGGTCTGCGAGAA-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'-TCCTTCCCGTTCTCCAGGTA-3'(SEQ ID NO:44)

HLA-K_F：5'-GGTATGAACAGCACGCCAAC-3'(SEQ ID NO:45)

HLA-K_R：5'-GCGTCTTGTGTTCCCTGGTA-3'(SEQ ID NO:46)

HLA-G_F：5'-ACCCTCTACCTGGGAGAACC-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'-TGCCGTCCAGTAACCAATC-3'(SEQ ID NO:50)

RAB11FIP4_F：5'-CGAGGGGAGGGCAAATTGAGT-3'(SEQ ID NO:51)

RAB11FIP4_R：5'-GAAGAAGGGACAAGGGGTGG-3'(SEQ ID NO:52)

CHFR_F：5'-GAGCTTTGATGGCAGAGTGTTA-3'(SEQ ID NO:53)

CHFR_R：5'-CTGGGAGCATGCATTTGTGAGA-3'(SEQ ID NO:54)

PNCK_F：5'-CTGTTGGCAGGTGAACCTCT-3'(SEQ ID NO:55)

PNCK_R：5'-CTGGGAAGGCTTGTCTCCTG-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'-GGGAACTCTCCCTCTGGTCT-3'(SEQ ID NO:60)

NPPA_F：5'-GAGCTTCTTGCATTGGTCCCT-3'(SEQ ID NO:61)

NPPA_R：5'-TCTGATCGATCTGCCCTCCT-3'(SEQ ID NO:62)

SYBR-based qPCR primers:

AFP_F：5'-AAATGCGTTTCTCGTTGCTT-3'(SEQ ID NO:63)

AFP_R：5'-GCCACAGGCCAATAGTTTGT-3'(SEQ ID NO:64)

SOX17_F：5'-CTCTGCCTCCTCCACGAA-3'(SEQ ID NO:65)

SOX17_R：5'-CAGAATCCAGACCTGCACAA-3'(SEQ ID NO:66)

BRACHYURY_F：5'-AATTGGTCCAGCCTTGGAAT-3'(SEQ ID NO:67)

BRACHYURY_R：5'-CGTTGCTCACAGACCACA-3'(SEQ ID NO:68)

FLK1_F：5'-TGATCGGAAATGACACTGGA-3'(SEQ ID NO:69)

FLK1_R：5'-CACGACTCCATGTTGGTCAC-3'(SEQ ID NO:70)

MAP2_F：5'-CAGGTGGCGGACGTGTGAAAATTGAGAGTG-3'(SEQ ID NO:71)

MAP2_R：5'-CACGCTGGATCTGCCTGGGGACTGTG-3'(SEQ ID NO:72)

PAX6_F：5'-GTCCATCTTTGCTTGGGAAA-3'(SEQ ID NO:73)

PAX6_R：5'-TAGCCAGGTTGCGAAGAACT-3'(SEQ ID NO:74)

TaqMan gene expression assay:

HLA-E:Hs03045171_m1

CD8:Hs00233520_m1

IL-2:Hs00174114_m1

RPLP0 (internal control): hs99999902_ m1

FACS antibodies

alpha-HLA-A2 (PE conjugated), clone BB7.2, Biolegend, Cat #343305

alpha-HLA-ABC (PE conjugated), clone W6/32, Biolegend, Cat #311406

alpha-HLA-E (PE conjugated), clone 3D12, Biolegend, Cat #342603

alpha-HLA-G (PE conjugated), clone MEM-G/9, Abcam, Cat # ab24384

alpha-HLA-DR (APC-conjugated), clone MEM-12, ThermoFisher Scientific, Cat # MA1-10347

alpha-B2M (APC conjugated), clone 2M2, Biolegend, Cat #316311

alpha-PD-L1 (APC conjugated), clone 29E.2A3, Biolegend, Cat #329708

alpha-PD-1 (APC conjugated), clone EH12.2H7, Biolegend, Cat #329908

alpha-CD 3(APC conjugated), clone UCHT1, Biolegend, Cat #300412

alpha-CD 3(Pacific blue TM conjugated), clone UCHT1, Biolegend, Cat #300418

alpha-CD 4(PE/Cy7 conjugated), clone RPA-T4, Biolegend, Cat #300511

alpha-CD 8(PE conjugated), clone SK1, Biolegend, Cat #344705

α-CD25(Alexa

700 conjugation), clone M-A251, Biolegend, Cat #356117

alpha-CD 47(PE conjugated), clone CC2C6, Biolegend, Cat #323108

alpha-CD 56(PE conjugated), clone HCD56, Biolegend, Cat #318306

α-CD69(Alexa

647 conjugate), clone FN50, Biolegend, Cat #310918

alpha-CD 107a (APC conjugated), clone H4A3, Biolegend, Cat #328620

alpha-CD 144(PE conjugated), clone 55-7H1, BD Biosciences, Cat #560410 isoform

Isoform 1: mouse IgG2b, kappa isotype control (APC-conjugated), Biolegend, Cat #400322

Isoform 2: mouse IgG2b, kappa isotype control (PE conjugated), Biolegend, Cat #401208

Isoform 3: mouse IgG2a, kappa isotype control (PE conjugated), Biolegend, Cat #400214

Immunofluorescent antibodies

α-OCT4，Abcam，Cat#ab19857

α-NANOG，Abcam，Cat#ab21624

α-SSEA3，Millipore，Cat#MAB4303

α-SSEA4，Millipore，Cat#MAB4304

α-TRA-1-60，Millipore，Cat#MAB4360

Donkey anti-rabbit IgG (H + L)Secondary antibody, Alexa

488 conjugate, Life Technologies, Cat # A-21206

Donkey anti-mouse IgG (H + L) secondary antibody, Alexa

488 conjugate, Life Technologies, Cat # A-2120

Goat anti-mouse IgM heavy chain Secondary antibody, Alexa

555 conjugate, Life Technologies, Cat # A-21426

Human ES cell culture, electroporation and drug selection

HUES8 cells (Cowan et al, 2004) were grown on Geltrex (Life Technologies) pre-coated plates and cultured in mTeSR1 (StemShell Technologies) supplemented with penicillin/streptomycin. For passage, cells were dissociated with Gentle Cell Dissociation Reagent (StemCell Technologies) for 5-10min and re-inoculated with RevitaCell^TM(ThermoFisher Scientific) in fresh medium. For electroporation, HUES8 cells were dissociated into single cells as described previously (Peters et al, 2013) and 1000 million cells were electroporated with 50. mu.g pCas9_ GFP (Addgene #44719) and a total of 50. mu.g gRNA plasmid for gene knock-out. For knock-in of the gene into the AAVS1 locus, cells were electroporated with 50 μ g pCas9_ GFP, 25 μ g gRNA _ AAVS1-T2(addge #41818) and 40 μ g double stranded donor plasmid. For gene knockout purposes, cells were harvested 48 hours after electroporation. GFP expressing cells were enriched by FACS (FACSAria II, BD Biosciences) and supplemented with RevitaCell^TMWas re-seeded at 15000 cells/plate on 10cm tissue culture plates to allow single cell colonies to form. Alternatively, for knock-in, cells 48 hours after electroporation were selected by 2. mu.g/ml blasticidin (ThermoFisher Scientific) for 5 days. The cell colonies were then manually picked and expanded.

CRISPR/Cas9 genome editing

500 base pairs of each region upstream or downstream of HLA-A/B/C were amplified from HUES8 or HEK293T cells and Sanger sequencing (Genewiz) was performed. Sequences conserved between the two cell lines were selected as reference sequences and sgrnas were designed using CRISPR design tools developed by the Massachusetts Institute of Technology (MIT) tension (fenghang) laboratory (available at: CRISPR. MIT. edu) and CCTop (Stemmer et al, 2015). The top-ranked sgRNA was picked and cloned into a gRNA expression vector (Addgene # 41824). The gRNA plasmid was then transfected into HEK293T cells, genomic DNA was extracted and the PCR amplicons covering the cleavage site were analyzed for on-target efficiency by TIDE (available at TIDE. nki. nl). The single guide RNA with the highest on-target activity was used for genome editing in HUES8 cells. To construct knock-in donor plasmids, the ORFs for PD-L1, HLA-G and CD47 were cloned separately and Gibson was used

(New England BioLab) was ligated by 2A sequence. After excising the Flpe-ERT2, the 3-in-1 (3-in-1) cassette was inserted between the Sal I and Mlu I restriction sites in the AAVS 1-Blastidine-CAG-Flpe-ERT 2 plasmid (Addge # 68461). Details of human ESC genome editing were previously described (Peters et al, 2013).

Generation of HLA knock-out (KO) cell lines

Briefly, to knock out the adjacent HLA-B/-C genes, a total of four sgrnas were co-electroporated into wild-type (WT) HUES8, along with the Cas9 expression plasmid. Homozygous knockout clones (HLA-B/-C) were then screened using the primers shown in FIG. 6A^-/-Efficiency: 1.56%). Hybrid knockout clones (HLA-B/-C) were also observed^+/-Efficiency: 7.8%). The elimination of HLA-B/-C mRNA expression of homozygous clones was further verified by RT-PCR and normal Karyotype was confirmed by nCounter Human Karyotype Assay (data not shown). Finally, one karyotypically normal clone was selected for further targeting of HLA-A and CIITA genes in one electroporation. PCR using the primers shown in FIG. 6B and flow cytometry using α -HLA-A2 antibody were performed to screen HLA-A knockout clones. The primers shown in FIG. 6E and Sanger sequencing were performed toCIITA knockout clones were identified. Therefore, only heterozygous clones (HLA-A) were observed after the first round of HLAA/CIITA targeting^+/-CIITA^+/indel)(HLA-A^+/-Efficiency: 3.68%). Thus, another round of HLA-a/CIITA sgRNA electroporation was applied to one karyotype normal heterozygous clone and the same screening strategy was used. Finally, a homozygous clone (HLA-A) was generated^-/-CIITA^indel/indel) However, FACS analysis showed that this clone was a mixed clone which still retained 1% of HLA-A⁺A cell. After subcloning, pure homozygous clones (HLA knock-out, KO) were obtained.

Karyotyping analysis

Karyotype G banding was performed by Cell Line Genetics.

Directed differentiation into three germ layers

WT and Gene-edited HuES8 cell line were made according to STEMdiff^TMThe monolayer-based approach of the Trilinkage Differentiation Kit (StemShell Technologies) differentiates into ectoderm, mesoderm and endoderm.

Differentiation into endothelial cells and vascular smooth muscle cells

Human Endothelial Cells (ECs) and Vascular Smooth Muscle Cells (VSMCs) were differentiated according to published protocols (Patsch et al, 2015). Briefly, for EC differentiation, ESCs were inoculated in N2B27 medium supplemented with 8uM CHIR99021(Cayman Chemical) and 25ng/ml BMP4(Peprotech) for 3 days to induce lateral mesoderm. EC was then induced by replacing the medium with StemPro-34 supplemented with 200ng/ml VEGF (Peprotech) and 2 μ M forskolin (Abcam) for 2 days. The cells were then enriched for CD144 using MACS cell isolation (Miltenyi Biotec)⁺A cell. In supplement with EGM^TM-2BulletKit^TM(Lonza) EBM ^TM2 in CD144⁺Cells were seeded on fibronectin (Corning) coated plates for further differentiation for at least 7 days. For VSMC differentiation, ESCs were inoculated in the same medium as EC differentiation for 3 days. On days 4 and 5, the medium was changed to N2B27 supplemented with 12.5ng/ml PDGF-BB (Peprotech) and 12.5ng/ml Activin A (Cell Guidance Systems). From day 6, cells were dissociated and supplemented with a Smooth Muscle Growth supplement (Smooth Muscle Growth Supp)lemment) (ThermoFisher Scientific) was seeded in medium 231 on gelatin-coated petri dishes for further differentiation.

Isolation and culture of human primary immune cells

Blood was obtained from a healthy, de-identified donor (leukopaks) from the Jackson Transfusion Center at the General Hospital of boston, Massachusetts General Hospital (Massachusetts Hospital). Human primary T cells, NK cells or CD14⁺The monocytes were individually passed through a negative selection kit (RosetteSep)^TM Human T Cell Enrichment Cocktail、RosetteSep^TMHuman NK Cell Enrichment Cocktail and rosetteSep^TMHuman monoclonal approach Cocktail, StemCell Technologies). Isolated T cells were cultured in X-VIVO 10(Lonza) medium supplemented with 5% human AB serum (Valley Biomedical), 5% fetal bovine serum, 1% penicillin/streptomycin, GlutaMAX, MEM nonessential amino acids (ThermoFisher Scientific), and 20U/ml IL-2 (Peprotech). Isolated NK cells were cultured in L-glutamine containing RPMI 1640(Corning) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The isolated monocytes were differentiated into macrophages in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 25-50ng/ml M-csf (peprotech).

Flow cytometry

PBS containing 1% Fetal Bovine Serum (FBS) was used as wash and stain buffer; PBS containing 4% FBS was used as blocking 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 were washed once and blocked with blocking buffer on ice for 20 min. In FACSCalibur^TMOr LSR II (BD Biosciences), cells were stained with antibody on ice for 30-60min and washed twice. Data were rendered using FlowJo software (BD).

In vitro T cell proliferation assay

When VSMC is used, cells are first treated with mitomycin (Fisher Scientific). 10 million ECs or VSMCs were inoculated onto 24-well plates and treated with IFN γ (100ng/ml) for 48 hours prior to assay. On day 0 of co-incubation, as preparedCellTrace for manufacturer's instruction^TMCfse (thermofisher scientific) labeled isolated CD3+ T cells. Adherent EC or VSMC were washed twice with PBS and then co-incubated with 500k CFSE labeled T cells for 5 days in T cell culture medium supplemented with 20U/ml IL-2. T cell staining was then performed with anti-CD 3/4/8 antibody before analyzing CFSE intensity on LSR II. T cells cultured for 5 days without target cells were used as negative controls. Using Dynabeads^TMT cells treated for 5 days with Human T-Activator CD3/CD28 beads (ThermoFisher Scientific) served as positive controls.

In vitro T cell activation assay and cytokine secretion assay

ESC-derived ECs were used as target cells. The co-culture conditions were the same as in the T cell proliferation assay, except that T cells were not labeled. After 5 days of co-culture, T cells were stained for T cell activation markers and then analyzed on LSR II. For multiple secreted cytokine quantification, supernatants were collected and analyzed by custom made MSD U-PLEX Platform (Meso Scale Discovery) according to manufacturer's instructions. T cells or target cells cultured for 5 days were used as negative controls. Using Dynabeads^TMHuman T-Activator CD3/CD28 beads (ThermoFisher Scientific) activated 5 days of T cells served as positive controls. Background activation was assessed from EC or VSMC using T cells incubated with conditioned media. Conditioned media were prepared as described above.

In vitro T/NK cell killing assay

ESC-derived VSMC were used as target cells. For the T cell killing assay, the co-incubation conditions were the same as for the T cell activation assay. For NK cell killing assays, 40K VSMC and NK cells were co-incubated for 20 hours in 200 μ l NK cell medium in 96-well U-bottoms at the indicated effector/target ratio before collecting the supernatant. After co-incubation, the supernatant was collected and passed through Pierce as per manufacturer's instructions^TMLDH cytoxicity Assay Kit (ThermoFisher Scientific) was analyzed. T cell culture medium or NK cell culture medium (RPMI-10) was used as background control. Separately cultured T/NK cells or separately cultured target cells were used as controls for spontaneous LDH release. Target cells lysed at the endpoint were used as maximum LDH release.

Pre-sensitization of allogeneic human CD8+ T cells

Using RosetteSep^TM Human CD8⁺Isolation of human Primary CD8 by T Cell Enrichment Cocktail (StemCell Technologies)⁺T cells and pre-primed with HUES 8-derived embryoid bodies as described previously (Gornaluse et al, 2017). Briefly, embryoid bodies were induced in suspension for 5 days, followed by adherent culture for 4 more days. Then CD8⁺T cells were co-cultured with adherent embryoid body cells for pre-sensitization. Extracellular matrix from a heterogeneous source (such as gelatin) is avoided in this process to prevent non-specific T cell activation.

In vivo T cell recall response assay

All Animal experiments were performed according to the regulations of the Harvard University Committee for International Animal Care and Use Committee. No randomization is used. All procedures were performed blind. Male immunodeficient SCID Beige mice (Taconc) 8-10 weeks old were used to form teratomas. 200 ten thousand HUES8 cells were packed in a clot and the clot was inserted subcutaneously on each side of the SCID Beige mice. Teratoma size was measured weekly by calipers after the teratoma became accessible. Between 4 and 6 weeks after hESC transplantation, 100 million pre-sensitized allogeneic human CD8 were administered⁺T cells were injected into mice via tail vein. Teratoma size was measured on days 2, 5 and 7 after T cell injection; teratoma size was also measured 2 days before T cell injection. On day 8 post-injection, teratomas were harvested and purified by qPCR and hematoxylin and eosin (H)&E) Staining for analysis. Two allogeneic CD8 were used under identical experimental conditions⁺T cell donors and results were pooled in this study. Histology was performed by the histological core of the Harvard Stem Cell Institute (Harvard Stem Cell Institute).

In vitro NK cell degranulation assay

24 hours prior to assay, 30 million adherent ESC derived VSMCs were seeded in 24-well plates. The following day, VSMC were washed once with PBS and then with 100K freshly isolated NK cells supplemented with α -CD107a APC (Biolegened) and eBioscience^TM Protein Transport Inhibitor Cocktail(ThermoFisher Scientific) in NK cell medium. After NK cells were added to the wells, the plates were centrifuged at 2,000rpm for 5min to achieve sufficient effector-target contact. After 20h of co-incubation, NK cells were stained with alpha-CD 56 PE (Biolegend) and then in FACSCalibur^TMCell surface expression of CD107a was analyzed as above. NK cell cultures without target cells were used as negative controls. NK cells treated with Cell Activation Cocktail (without Brefeldin A), which includes PMA (phorbol 12-myristate-13-acetate) and ionomycin, were used as positive controls for degranulation.

In vitro macrophage phagocytosis assay

Using RosetteSep^TMHuman monoclonal enzyme cocktails (StemCell Technologies) isolate monocytes from donor blood via negative selection. Monocytes were seeded in serum-free medium for attachment and maturation to macrophages in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and 25ng/ml M-csf (peprotech) for one to three weeks. Two days prior to the assay, macrophages were re-seeded into a 96-well test board (ibidi) at a density of 100K/well. For the assay, differentiated VSMCs were pretreated with 200nM staurosporine (Sigma) for 1.5 hours for the "STS treated" group. VSMC were dissociated and labeled with pHrodo-Red (IncuCyte) for 1h at 37 ℃. Thirty thousand labeled VSMCs were added to each macrophage containing well and the co-incubated cultures were immediately transferred to the celldisscover 7 live cell imaging platform (Zeiss). One image of red fluorescence emission after phagocytosis per well was taken every 20min for 6 hours. The total integrated intensity (mean fluorescence intensity total area) of each image was analyzed using ZEN imaging software (Zeiss). pHrodo-Red⁺The particles represent phagosomes within macrophages that have phagocytosed VSMCs.

Generation of CD 47-/-and B2M-/-HUES8 cell lines

The following four CRISPR sgrnas were used to target the first coding exon of CD47 in HUES8 cells.

5'-gGTCCTGCCTGTAACGGCGG-3'(SEQ ID NO:75)

5'-gGACCGCCGCCGCGCGTCAC-3'(SEQ ID NO:76)

5'-gCAGCAACAGCGCGCCTACC-3'(SEQ ID NO:77)

5'-gTTCGCCCCCGCGGGCGTGT-3'(SEQ ID NO:78)

Cells were stained with anti-CD 47 antibody (Clone CC2C6) 72 hours after electroporation and CD47 negative cells were isolated using FACS Aria (BD). Single cell derived colonies were obtained as described previously (Peters et al, 2013) and subsequently confirmed for loss of CD47 expression by FACS analysis. Similarly, a Beta-2-microgludulin (B2M) deficient HUES8 cell line was generated using the following sgrnas:

5’-gCTACTCTCTCTTTCTGGCC-3’。(SEQ ID NO:79)

lentiviral transduction

A doxycycline-inducible lentiviral Gateway vector (Invitrogen) containing the PD-L1 ORF was constructed by PCR amplification. The PD-L1 expressing lentivirus was packaged by transfecting HEK293T cells with PD-L1 expression vector and packaging plasmids pMDL, pVSVG and pREV. The lentiviral particle-containing medium was collected 48 hours post-transfection and used to transduce VSMCs along with lentiviral particles encoding doxycycline-binding transactivator rtTA. After 24 hours, VSMC were treated with doxycycline (10 μ g/ml) to induce PD-L1 expression, as verified by FACS. T cell proliferation was evaluated against VSMC overexpressing PD-L1 as described above, except for 7 days of co-incubation and the presence of doxycycline throughout the co-incubation. No effect on T cell proliferation was observed with doxycycline addition.

Immunofluorescence

PBS containing 0.05% Tween-20 was a 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 with 4% Donkey Serum (Donkey Serum) (Jackson ImmunoResearch Laboratories) overnight at 4 ℃ and incubated with the appropriate primary antibody diluted in blocking buffer for 1 hour at room temperature. The cells were then incubated with Alexa

488-or Alexa

555 conjugated secondary antibodies (Life Technologies). Cells were washed and nuclei stained with Hoechst. The images were visualized with a Nikon inverted microscope.

RNA isolation, cDNA Synthesis and qPCR

RNA was extracted using a TRIzol Reagent (ThermoFisher Scientific) according to the manufacturer's instructions. cDNA synthesis was performed using SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific) according to the manufacturer's protocol. SYBR green-based or TaqMan-based qPCR was performed and relative quantitation was determined using the QuantStaudio 12k Flex System (ThermoFisher Scientific) and then by the comparative Ct method (2)^-ΔΔCt) To calculate expression relative to the respective internal control.

Next Generation Sequencing (NGS) -based off-target analysis

The off-target sites were predicted using CCTOP (Stemmer et al, 2015). Bait design, genomic DNA enrichment (library preparation) and NGS use

Custom target capture kits were performed by Arbor Biosciences. Briefly, for each of the 648 predicted off-target sites, five RNA decoys were designed across each off-target site and placed once every 26bp, covering a window of 181-. After extraction of genomic DNA from WT and three engineered hPSC lines, biotinylated RNA decoys were hybridized to the corresponding denatured genomic DNA libraries. Subsequently, the RNA-gDNA hybrid was bound to streptavidin-coated beads and the non-specific bonds were washed away. The remaining gDNA library was amplified and sequenced by double-ended NGS using novaseq (illumina).

Unless stated later, genome editing events were quantified by crispresospooled from the crispresoso suite (1.0.13 th edition) at default settings (Pinello et al, 2016). Briefly, for each of the four libraries, reads with a minimum single base pair score (phred33) greater than 25 were selected and aligned to a ± 100bp window around each gRNA off-target site in the human genome (hg 38). Fewer than 5 aligned reads of any library were filtered out (═ 3). The percentage of reads with altered sequences (insertions, deletions and substitutions) compared to hg38 at each off-target site from each library was calculated by the program. If the percentage of reads with altered sequences was found to be >0 in WT and in all three engineered lines, the sequences were further examined. If the sequences of all three engineered lines match the WT sequence, they are classified as SNP/PM; however, if the sequence of the engineered cell lines deviates from the WT sequence, they are identified as editing events. Polymorphism (PM) represents a small deletion/insertion rather than the Single Nucleotide Polymorphism (SNP) deviating from hg38 that has been observed in WT hpscs.

Statistical analysis

Graphs were generated using Prism 7(Graphpad) and statistically analyzed.

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Claims (76)

1. A stem cell comprising reduced expression of MHC-I and MHC-II human leukocyte antigens relative to a wild-type stem cell and increased expression of tolerogenic factors relative to a wild-type stem cell,

wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C,

wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR, and

wherein the tolerogenic factor is CD 47.

2. The stem cell of claim 1, wherein the decreased expression of the MHC-I human leukocyte antigens comprises a deletion of the MHC-I human leukocyte antigens from at least one allele of the cell.

3. The stem cell of claim 1 or claim 2, wherein the reduced expression of MHC-II human leukocyte antigens comprises introducing one or more indels into CIITA.

4. The stem cell of any one of claims 1-3, further comprising reduced expression of CIITA.

5. The stem cell of any one of claims 1-4, wherein the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell.

6. The stem cell of any one of claims 1-5, wherein the stem cell does not express HLA-A, HLA-B and HLA-C.

7. The stem cell of any one of claims 1-6, wherein the stem cell does not express HLA-DP, HLA-DQ, and HLA-DR.

8. The stem cell of any one of claims 1-7, wherein the stem cell does not express CIITA.

9. The stem cell of any one of claims 1-8, wherein the tolerogenic factors further comprise HLA-G and/or PD-L1.

10. A stem cell which does not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR and which expresses CD 47.

11. The stem cell of claim 10, wherein the cell is a CIITAindel/indel, HLA-A-/-, HLA-B-/-and HLA-C-/-stem cell.

12. A method of producing a hypoimmunogenic stem cell, the method comprising decreasing expression of MHC-I and MHC-II human leukocyte antigens of a stem cell and increasing expression of tolerogenic factors of the stem cell, thereby producing the hypoimmunogenic stem cell,

wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C,

wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR, and

wherein the tolerogenic factor is CD 47.

13. The method of claim 12, wherein reducing the expression of the MHC-I human leukocyte antigens comprises deleting the MHC-I human leukocyte antigens from at least one allele of the stem cells.

14. The method of claim 12 or claim 13, wherein reducing the expression of the MHC-II human leukocyte antigen comprises introducing one or more indels into CIITA.

15. The method of any one of claims 12-14, further comprising reducing expression of CIITA.

16. The method of any one of claims 12-15, wherein increasing expression of a tolerogenic factor comprises inserting the tolerogenic factor into a safe harbor locus of at least one allele of the stem cell.

17. The method of any one of claims 12-16, wherein the tolerogenic factors further comprise PD-L1 and/or HLA-G.

18. The method of any one of claims 12-17, wherein the hypoimmunogenic stem cells do not express HLA-A, HLA-B and HLA-C.

19. The method of any one of claims 12-18, wherein the low immunogenic stem cells do not express HLA-DP, HLA-DQ and HLA-DR.

20. The method of any one of claims 12-19, wherein the hypoimmunogenic stem cells do not express CIITA.

21. A stem cell comprising modulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors relative to a wild-type stem cell.

22. The stem cell of claim 21, wherein the one or more MHC-I human leukocyte antigens are selected from HLA-A, HLA-B and HLA-C.

23. The stem cell of claim 21 or 22, wherein the modulated expression of the one or more MHC-I human leukocyte antigens comprises a decrease in expression of the one or more MHC-I human leukocyte antigens.

24. The stem cell of any one of claims 21-23, wherein the one or more MHC-I human leukocyte antigens are deleted from the genome of the cell, thereby modulating expression of the one or more MHC-I human leukocyte antigens.

25. The stem cell of any one of claims 21-24, wherein the one or more MHC-II human leukocyte antigens are selected from HLA-DP, HLA-DQ and HLA-DR.

26. The stem cell of any one of claims 21-25, wherein the modulated expression of the one or more MHC-II human leukocyte antigens comprises a decrease in expression of the one or more MHC-II human leukocyte antigens.

27. The stem cell of any one of claims 21-26, wherein one or more indels are introduced into CIITA, thereby modulating expression of the one or more MHC-II human leukocyte antigens.

28. The stem cell of any one of claims 21-27, wherein the cell does not express HLA-A, HLA-B and HLA-C.

29. A stem according to any one of claims 21 to 28A cell, wherein the cell is HLA-a^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell.

30. The stem cell of any one of claims 21-29, wherein the one or more tolerogenic factors are selected from HLA-G, PD-L1 and CD 47.

31. The stem cell of any one of claims 21-30, wherein modulated expression of the one or more tolerogenic factors comprises increased expression of the one or more tolerogenic factors.

32. The stem cell of any one of claims 21-31, wherein the one or more tolerogenic factors are inserted into the AAVS1 safety harbor locus.

33. The stem cell of any one of claims 21-32, wherein HLA-G, PD-L1 and CD47 are inserted into the AAVS1 safe harbor locus.

34. The stem cell of any one of claims 21-33, wherein the one or more tolerogenic factors inhibit immune rejection.

35. The stem cell of any one of claims 21-34, wherein the stem cell is an embryonic stem cell.

36. The stem cell of any one of claims 21-34, wherein the stem cell is a pluripotent stem cell.

37. The stem cell of any one of claims 21-34, wherein the stem cell is less immunogenic.

38. The stem cell of any one of claims 21-34, wherein the stem cell is a human stem cell.

39. The stem cell of any one of claims 21-38, wherein the stem cell retains pluripotency.

40. The stem cell of any one of claims 21-39, wherein the stem cell retains differentiation potential.

41. The stem cell of any one of claims 21-40, wherein the stem cell exhibits a reduced T cell response.

42. The stem cell of any one of claims 21-41, wherein the stem cell exhibits protection from NK cell responses.

43. The stem cell of any one of claims 21-42, wherein the stem cell exhibits reduced macrophage phagocytosis.

44. A stem cell which does not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR.

45. The stem cell of claim 44, wherein the stem cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell.

46. The stem cell of claim 44 or 45, wherein the stem cell expresses the tolerogenic factors HLA-G, PD-L1 and CD 47.

47. The stem cell of claim 46, wherein the tolerogenic factor is inserted into the AAVS1 safety harbor locus.

48. The stem cell of claim 46 or 47, wherein the tolerogenic factor inhibits immune rejection.

49. The stem cell of claims 44-48, wherein the stem cell is an embryonic stem cell.

50. The stem cell of claims 44-48, wherein the stem cell is a pluripotent stem cell.

51. The stem cell of claims 44-48, wherein the stem cell is less immunogenic.

52. A method of producing a stem cell with reduced immunogenicity, the method comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of the stem cell relative to a wild-type stem cell, thereby producing the stem cell with reduced immunogenicity.

53. The method of claim 52, wherein the one or more MHC-I human leukocyte antigens are selected from HLA-A, HLA-B and HLA-C.

54. The method of claim 52 or 53, wherein the modulated expression of the one or more MHC-I human leukocyte antigens comprises a decrease in expression of the one or more MHC-I human leukocyte antigens.

55. The method of any one of claims 52-54, wherein the one or more MHC-I human leukocyte antigens are deleted from the genome of the stem cell, thereby modulating expression of the one or more MHC-I human leukocyte antigens.

56. The method of any one of claims 52-55, wherein the one or more MHC-II human leukocyte antigens are selected from HLA-DP, HLA-DQ and HLA-DR.

57. The method of any one of claims 52-56, wherein the modulated expression of the one or more MHC-II human leukocyte antigens comprises a decrease in expression of the one or more MHC-II human leukocyte antigens.

58. The method of any one of claims 52-57, wherein one or more indels are introduced into CIITA, thereby modulating expression of the one or more MHC-II human leukocyte antigens.

59. The method of any one of claims 52-58, wherein the hypoimmunogenic stem cells do not express HLA-A, HLA-B and HLA-C.

60. The method of any one of claims 52-59, wherein the hypoimmunogenic stem cell is HLA-A^-/-、HLA-B^-/-、HLA-C^-/-And CIITA^indel/indelA cell.

61. The method of any one of claims 52-60, wherein the one or more tolerogenic factors are selected from HLA-G, PD-L1 and CD 47.

62. The method of any one of claims 52-61, wherein the modulated expression of the one or more tolerogenic factors comprises increased expression of the one or more tolerogenic factors.

63. The method of any one of claims 52-62, wherein the one or more tolerogenic factors are inserted into the AAVS1 safety harbor locus.

64. The method of any one of claims 52-63, wherein HLA-G, PD-L1 and CD47 are inserted into the AAVS1 safety harbor locus.

65. The method of any one of claims 52-64, wherein the one or more tolerogenic factors inhibit immune rejection.

66. The method of any one of claims 52-65, wherein the hypoimmunogenic stem cells retain pluripotency.

67. The method of any one of claims 52-66, wherein the hypoimmunogenic stem cells retain differentiation potential.

68. The method of any one of claims 52-67, wherein the hypoimmunogenic stem cells exhibit a reduced T cell response.

69. The method of any one of claims 52-68, wherein the hypoimmunogenic stem cells exhibit protection from NK cell responses.

70. The method of any one of claims 52-69, wherein the hypoimmunogenic stem cells exhibit reduced macrophage phagocytosis.

71. The method of claim 52, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences SEQ ID NOs: 1-2, thereby editing the HLA-A gene to reduce or eliminate HLA-A surface expression and/or activity in the stem cell.

72. The method of claim 52, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences SEQ ID NOS 3-4, thereby editing the HLA-B gene to reduce or eliminate HLA-B surface expression and/or activity in the stem cell.

73. The method of claim 52, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences SEQ ID NOS 5-6, thereby editing an HLA-C gene to reduce or eliminate HLA-C surface expression and/or activity in the stem cell.

74. The method of claim 52, wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a ribonucleic acid having the sequence SEQ ID NO:7, thereby introducing an indel into CIITA to reduce or eliminate MHC-II human leukocyte antigen surface expression and/or activity in the stem cell.

75. A method of producing a stem cell with reduced immunogenicity, the method comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of the stem cell relative to a wild-type stem cell, thereby producing the stem cell with reduced immunogenicity,

wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having the sequences SEQ ID NOS: 1-2, thereby editing the HLA-A gene to reduce or eliminate HLA-A surface expression and/or activity in the stem cell,

wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acids having the sequences SEQ ID NOS: 3-4, thereby editing the HLA-B gene to reduce or eliminate HLA-B surface expression and/or activity in the stem cell,

wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third pair of ribonucleic acids having the sequences SEQ ID NOS: 5-6, thereby editing the HLA-C gene to reduce or eliminate HLA-C surface expression and/or activity in the stem cell, and

wherein the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a ribonucleic acid having the sequence SEQ ID NO. 7, thereby introducing an indel into CIITA to reduce or eliminate MHC-II human leukocyte antigen surface expression and/or activity in the stem cell.

76. A method of transplanting at least one hypoimmunogenic stem cell into a patient, wherein the hypoimmunogenic stem cell comprises modulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors, relative to a wild-type stem cell.

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