WO2020168317A2 - Cellules souches donnatrices universelles et méthodes associées - Google Patents

Cellules souches donnatrices universelles et méthodes associées Download PDF

Info

Publication number
WO2020168317A2
WO2020168317A2 PCT/US2020/018467 US2020018467W WO2020168317A2 WO 2020168317 A2 WO2020168317 A2 WO 2020168317A2 US 2020018467 W US2020018467 W US 2020018467W WO 2020168317 A2 WO2020168317 A2 WO 2020168317A2
Authority
WO
WIPO (PCT)
Prior art keywords
hla
stem cell
cell
mhc
expression
Prior art date
Application number
PCT/US2020/018467
Other languages
English (en)
Other versions
WO2020168317A8 (fr
WO2020168317A3 (fr
Inventor
Torsten B. Meissner
Leonardo M.R. FERREIRA
Jack L. Strominger
Chad A. COWAN
Original Assignee
President And Fellows Of Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CA3130398A priority Critical patent/CA3130398A1/fr
Priority to CN202080021860.7A priority patent/CN113906048A/zh
Priority to AU2020223192A priority patent/AU2020223192A1/en
Priority to SG11202108891QA priority patent/SG11202108891QA/en
Priority to JP2021547755A priority patent/JP2022526218A/ja
Priority to KR1020217029445A priority patent/KR20210128440A/ko
Priority to EA202192264A priority patent/EA202192264A1/ru
Priority to MX2021009842A priority patent/MX2021009842A/es
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority to EP20755906.3A priority patent/EP3924375A4/fr
Priority to BR112021016178A priority patent/BR112021016178A2/pt
Publication of WO2020168317A2 publication Critical patent/WO2020168317A2/fr
Publication of WO2020168317A3 publication Critical patent/WO2020168317A3/fr
Publication of WO2020168317A8 publication Critical patent/WO2020168317A8/fr
Priority to IL285619A priority patent/IL285619A/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Disclosed herein are efficient strategies to overcome immune rejection in cell- based transplantation therapies by the creation of universal donor stem cell lines.
  • 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 a wild-type stem cell.
  • the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C.
  • the modulated expression of the one or more MHC-I human leukocyte antigens comprises reduced expression of the one or more MHC-I human leukocyte antigens.
  • the one or more MHC-I human leukocyte antigens are deleted from the genome of the cell, thereby modulating the expression of the one or more MHC-I human leukocyte antigens.
  • the one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR.
  • the modulated expression of the one or more MHC-II human leukocyte antigens comprises reduced expression of the one or more MHC-II human leukocyte antigens.
  • one or more indels were introduced into CIITA, thereby modulating the expression of the one or more MHC-II human leukocyte antigens.
  • the cell does not express HLA-A, HLA-B, and HLA-C. In certain aspects, the cell is an HLA-A 7 , HLA-B 7 , HLA-C 7 , and CUT A mdel/mdel cell.
  • the one or more tolerogenic factors are selected from the group consisting of HLA-G, PD-L1, and CD47.
  • the modulated expression of the one or more tolerogenic factors comprises increased expression of the one or more tolerogenic factors.
  • the one or more tolerogenic factors are inserted into an AAVS1 safe harbor locus.
  • HLA-G, PD-L1, and CD47 are inserted into an AAVS1 safe harbor locus.
  • the one or more tolerogenic factors inhibit immune rejection.
  • the stem cell is an embryonic stem cell. In some aspects, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is hypoimmunogenic. In some aspects, the stem cell is a human stem cell.
  • the stem cell retains pluripotency. In some aspects, the stem cell retains differentiation potential. In some embodiments, the stem cell exhibits reduced T cell response. In some aspects, the stem cell exhibits protection from NK cell response. In some embodiments, the stem cell exhibits reduced macrophage engulfment. Also disclosed herein are stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.
  • the stem cell is a HLA-A 7 , HLA-B 7 , HLA-C 7 , and CIITA mdel/mdel cell.
  • the stem cell expresses tolerogenic factors HLA- G, PD-L1, and CD47.
  • the tolerogenic factors are inserted into an AAVS1 safe harbor locus. In certain aspects, the tolerogenic factors inhibit immune rejection.
  • the stem cell is an embryonic stem cell. In some aspects, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is hypoimmunogenic .
  • Disclosed herein are methods of preparing a hypoimmunogenic stem cell the method comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of a stem cell relative to a wild-type stem cell, thereby preparing the hypoimmunogenic stem cell.
  • the one or more MHC-I human leukocyte antigens are selected from the group consisting of HLA-A, HLA-B, and HLA-C.
  • the modulated expression of the one or more MHC-I human leukocyte antigens comprises reduced expression of the one or more MHC-I human leukocyte antigens.
  • the one or more MHC-I human leukocyte antigens are deleted from the genome of the stem cell, thereby modulating the expression of the one or more MHC-I human leukocyte antigens.
  • the one or more MHC-II human leukocyte antigens are selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR.
  • the modulated expression of the one or more MHC-II human leukocyte antigens comprises reduced expression of the one or more MHC-II human leukocyte antigens.
  • one or more indels were introduced into CIITA, thereby modulating the expression of the one or more MHC-II human leukocyte antigens.
  • the hypoimmunogenic stem cell does not express HLA-A, HLA-B, and HLA-C. In some embodiments, the hypoimmunogenic stem cell is an HLA-A 7 , HLA-B - 7 , HLA-C 7 , and CUT A indel/indel cell.
  • the one or more tolerogenic factors are selected from the group consisting of HLA-G, PD-L1, and CD47.
  • the modulated expression of the one or more tolerogenic factors comprises increased expression of the one or more tolerogenic factors.
  • the one or more tolerogenic factors are inserted into an AAVS1 safe harbor locus. In some aspects, HLA-G, PD-L1, and CD47 are inserted into an AAVS1 safe harbor locus. In some embodiments, the one or more tolerogenic factors inhibit immune rejection.
  • the hypoimmunogenic stem cell retains pluripotency.
  • the hypoimmunogenic stem cell retains differentiation potential. In some embodiments, the hypoimmunogenic stem cell exhibits reduced T cell response. In some aspects, the hypoimmunogenic stem cell exhibits protection from NK cell response. In some embodiments, the hypoimmunogenic stem cell exhibits reduced macrophage engulfment.
  • 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.
  • 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: 3-4, thereby editing the HLA-B gene to reduce or eliminate HLA-B surface expression and/or activity in the stem cell.
  • 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: 5-6, thereby editing the HLA-C gene to reduce or eliminate HLA-C surface expression and/or activity in the stem cell.
  • the stem cell is contacted with a Cas protein or a nucleic acid sequence encoding the Cas protein and a ribonucleic acid having sequence SEQ ID NO: 7, thereby introducing indels into CIITA to reduce or eliminate MHC-II human leukocyte antigens surface expression and/or activity in the stem cell.
  • Also disclosed herein are methods of preparing a hypoimmunogenic stem cell the method comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors of a stem cell relative to a wild-type stem cell, thereby preparing the hypoimmunogenic 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
  • 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.
  • the stem cells may comprise reduced expression of MHC-I and MHC-II human leukocyte antigens relative to a wild-type stem cell and increased expression of a tolerogenic factor 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 CD47.
  • the reduced expression of the MHC-I human leukocyte antigens comprises the MHC-I human leukocyte antigens being deleted from at least one allele of the cell. In some embodiments, the reduced expression of the MHC-II human leukocyte antigens comprises one or more indels being introduced 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.
  • the stem cell does not express HLA-A, HLA-B, and HLA-C. In some embodiments, the stem cell does not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the stem cell does not express CIITA. In some embodiments, the tolerogenic factor further comprises HLA-G and/or PD-L1.
  • stem cells that do not express HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR and expresses CD47.
  • the cell is a CIITAindel/indel, HLA-A-/-, HLA-B-/-, and HLA-C-/- stem cell.
  • the methods may comprise decreasing expression of MHC-I and MHC-II human leukocyte antigens of a stem cell and increasing expression of a tolerogenic factor of the stem cell, thereby preparing 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 CD47.
  • reducing expression of the MHC-I human leukocyte antigens comprises deleting the MHC-I human leukocyte antigens from at least one allele of the stem cell. In some embodiments, reducing expression of the MHC-II human leukocyte antigens comprises introducing one or more indels into CIITA. In some embodiments, the methods further comprise reducing expression of CIITA. In some embodiments, 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.
  • the tolerogenic factor further comprises PD-L1 and/or HLA-G.
  • the hypoimmunogenic stem cell does not express HLA-A, HLA-B, and HLA-C.
  • the hypoimmunogenic stem cell does not express HLA-DP, HLA-DQ, and HLA-DR.
  • the hypoimmunogenic stem cell does not express CIITA.
  • FIGS. 1A-1J demonstrate genome editing ablates polymorphic HLA-A/-B/-C and HLA class II expression and enables expression of immunomodulatory factors from AAVS1 safe harbor locus.
  • FIG. 1 A provides a schematic representation of HLA- B and HLA-C CRISPR/Cas9 knockout strategy. Each pair of scissors represents two sgRNAs. Purple, red, and green arrows indicate primers used for PCR screening.
  • FIG. IB provides a schematic representation of HLA- A knockout strategy. Each pair of scissors represents one sgRNA. Yellow arrows show primers used for PCR screening.
  • FIG. 1C provides FACS contour plots demonstrating successful ablation of HLA-A/B/C in HUES 8. Wild-type (WT) or HLA-A/B/C knockout (KO) cells were treated with IENg for 48 hrs before staining with the indicated antibodies.
  • FIG. ID shows targeting strategy of CIITA locus. Blue arrows indicate primers used for PCR and Sanger sequencing.
  • FIG. IE shows HLA-DR mean fluorescence intensity (MFI) in differentiated CD144 + WT and KO ECs.
  • FIG. 1G shows knock-in strategy of immune modulatory molecules. Scissors represent the sgRNA targeting the AAVS1 locus. Black and gray arrows indicate primers used for PCR screening.
  • FIG. 1H shows PD-L1 and HLA-G expression in KI-PHC cells.
  • FIG. II shows CD47 expression in KI-PHC cells. MFIs relative to WT cells are indicated on the right of histograms.
  • FIG. 1J shows PD-L1 and CD47 expression in KI-PC cells.
  • FIGS. 2A-2E demonstrate KO and KI cell lines retain pluripotency and differentiation potential.
  • FIG. 2A shows immunofluorescence indicating that pluripotency markers were expressed by WT, KO, KI-PHC, and KI-PC human pluripotent stem cells (hPSCs). Scale bars, 200 pm.
  • FIG. 2B shows qRT-PCR was carried out to survey trilineage markers after WT, KO, KI-PHC, and KI-PC hPSCs were differentiated into the indicated three germ layers. Relative quantification was normalized to each gene level in unmodified hPSCs.
  • FIG. 2A shows immunofluorescence indicating that pluripotency markers were expressed by WT, KO, KI-PHC, and KI-PC human pluripotent stem cells (hPSCs). Scale bars, 200 pm.
  • FIG. 2B shows qRT-PCR was carried out to survey trilineage markers after WT, KO, KI-PHC, and
  • FIG. 2C shows G-banding of chromosomes in KO, KI-PHC, and KI-PC cell lines demonstrated normal karyotypes after successive rounds of genome engineering.
  • FIG. 2D provides a table showing the PCR-based analyses of exonic off-target sites in engineered hPSC lines.
  • FIG. 2E shows target capture sequencing results showing the % reads with altered sequence at off-target sites in WT and engineered hPSC lines. Black circle, SNP/polymorphism (PM) site; red circle, edited off-target site; blue circle, CIITA on-target site as positive control.
  • PM SNP/polymorphism
  • FIGS. 3A-3D demonstrate reduced T cell activities against KO and KI-PHC cell lines in vitro.
  • T cells cultured alone were used as negative control; T cells activated with CD3/CD28 beads served as positive controls. Paired one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ⁇ s.e.m.; * p ⁇ 0.05; ** p ⁇ 0.01.
  • the same negative and positive controls were used as in A. Paired one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ⁇ s.e.m.; ** p ⁇ 0.01; *** p ⁇ 0.001 ; **** p ⁇ 0.0001.
  • 3C provides bar graphs of IFNy (left panel) and IL-10 (right panel) concentration in the medium following co-culture of WT, KO, or KI ECs with CD3 + T cells from one representative donor. Spontaneous release from T cells alone were used as negative controls. Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ⁇ s.d.; ** p ⁇ 0.01 ; *** p ⁇ 0.001.
  • FIG. 3D provides a bar graphs of IFNy (left panel) and IL-10 (right panel) concentration in the medium following co-culture of WT, KO, or KI ECs with CD3 + T cells from one representative donor. Spontaneous release from T cells alone were used as negative controls. Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ⁇ s.d.; ** p ⁇ 0.01 ; *** p ⁇ 0.001.
  • FIG. 3D provides a bar graphs of IFNy (left panel) and
  • FIGS. 4A-4E demonstrate reduced T cell responses against KO and KI cell lines in vivo.
  • FIG. 4A provides a schematic describing the pre- sensitization of allogeneic CD8 + T cells and the workflow of in vivo T cell recall response assay.
  • FIG. 4B shows percentage of increased teratoma volume on day 5 or 7 post T cell injection compared to day 0.
  • FIG. 4C shows percentage of increased teratoma volume on day 0 of T cell injection compared to 2 days pre injection.
  • the expression was normalized to RPLP0. Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ⁇ s.e.m.; * p ⁇ 0.05; ** p ⁇ 0.01.
  • FIG. 4E shows representative hematoxylin and eosin (H&E) staining of WT, KO, KI-PHC, and KI-PC teratomas harvested on day 8 post T cell injection. The black arrows indicate the sites of T cell infiltration. Scale bars, 100 pm.
  • FIGS. 5A-5D demonstrate KI cell lines are protected from NK cell and macrophage responses.
  • LDH release assay was performed and the % NK cytotoxicity was calculated as specific lysis of NK cell-killed VSMCs relative to maximum cell lysis.
  • pHrodo-red-labelled VSMCs of indicated genotypes that were pretreated (right panel) or not pretreated (left panel) with Staurosporine (STS) were co-incubated with monocyte-derived macrophages for 6 hrs. Images were acquired every 20 min using Celldiscover 7 live cell imaging system. Total integrated fluorescence intensity of pHrodored+ phagosomes per image was analyzed. Data are mean ⁇ s.e.m.
  • FIGS. 6A-6H demonstrate genome editing ablates polymorphic HLA-A/-B/-C and HLA class II expression and enables expression of immunomodulatory factors from AAVS1 safe harbor locus.
  • FIG. 6A shows PCR confirmation of HLA-B/-C knockout using primers shown in FIG. 1A.
  • FIG. 6B shows PCR confirmation of HLA- A knockout using primers shown in FIG. IB.
  • FIG. 6C shows PCR products using the primers flanking the CIITA cutting site.
  • FIG. 6D shows Sanger sequencing reveals that in the KO cell line, 1 bp (shown in red) was inserted on one CIITA allele and 12 bp (shown as dashes) were deleted from the other allele.
  • FIG. 6A shows PCR confirmation of HLA-B/-C knockout using primers shown in FIG. 1A.
  • FIG. 6B shows PCR confirmation of HLA- A knockout using primers shown in FIG. IB.
  • FIG. 6E shows CD144 expression in differentiated WT and KO endothelial cells (ECs).
  • FIG. 6F shows workflow of generating KO and KI ES cell lines.
  • FIG. 6G shows PCR confirmation of knock-in of the KI-PHC/KI-PC constructs using primers shown in FIG. 1G.
  • FIGS. 7A-7H demonstrate genome editing ablates polymorphic HLA-A/-B/-C and HLA class II expression and enables expression of immunomodulatory factors from AAVSl safe harbor locus.
  • FIG. 7A shows CD47 expression in WT and KI-PC ES cells. MFIs relative to WT cells are given on the right of the histograms.
  • FIG. 7B shows HLA-A2 expression in WT, KI-PHC, and KI-PC ES cells post IFNy treatment confirming the ablation of classical HLA class la molecules in the KI cell lines.
  • FIG. 7C shows CD144 expression in differentiated WT, KI-PHC, and KI-PC ECs.
  • FIG. 7D shows HLA-DR mean fluorescence intensity (MFI) confirming the ablation of HLA class II in differentiated KI-PHC and KI-PC ECs. HLA-DR expression was analyzed on CD144 + cells.
  • FIG. 7E shows CD140b expression in differentiated WT, KO, KI-PHC, and KI-PC VSMCs.
  • FIG. 7F provides contour plots showing the expression of PD-L1 and HLA-G in differentiated WT and KI-PHC VSMCs (upper left panel). CD47 expression in differentiated WT and KI-PHC VSMCs (upper right panel). Contour plots showing the expression of PD-L1 and CD47 in differentiated WT and KI-PC VSMCs (lower left panel). CD47 expression in differentiated WT and KI-PC VSMCs (lower right panel).
  • FIG. 7G shows HLA-E expression in
  • FIG. 7H shows relative HLA-E mRNA expression in differentiated WT, B2M _/ , KO, and KI-PHC VSMCs with or without IFN-g stimulation.
  • FIG. 8 provides sequencing chromatograms of predicted exonic off-target sites in gene-modified hPSC lines and in parental WT cells.
  • FIG. 9 shows Sequence inspection from NGS showing editing at off-target sites in engineered hPSC lines, and the SNP/polymorphic sites observed in engineered lines as well as WT cells.
  • FIGS. 10A-10D demonstrate reduced T cell activities against KO and KI-PHC cell lines.
  • FIG. 10A shows gating strategy used in T cell proliferation and activation assays.
  • FIG. 10B provides a T cell proliferation assay of one representative donor using WT, KO, and KI-PHC ECs as target cells.
  • CD3 + top panel
  • CD4 + top panel
  • CD8 + bottom panel
  • T cells cultured alone were used as negative control
  • T cells treated with CD3/CD28 beads served as positive control.
  • FIG. IOC shows doxycyc line-inducible PD-L1 expression in WT VSMCs.
  • T cells with reduced CFSE signal were quantified as proliferating cells.
  • T cells cultured alone served as negative control; T cells activated with CD3/CD28 beads were used as positive control. Paired two tailed t-test; Data are mean ⁇ s.e.m.; * p ⁇ 0.05; ns, no significance.
  • FIGS. 11A-11E demonstrate KI cell lines are protected from NK cell and macrophage responses.
  • FIG. 11A shows CD69 and PD-1 expression examined in pre and post priming of one representative CD8 + T donor.
  • FIG. 11B provides gating strategy of NK cell degranulation assay.
  • FIG. 11 C provides FACS contour plots of NK cell degranulation assay for one representative donor.
  • FIG. 11D shows CD47 MFI confirming the ablation of CD47 expression in differentiated CD47 _/ VSMCs.
  • FIG. 11E provides fluorescence images showing engulfed VSMCs pre-labeled with pHrodo-Red after 4h co-incubation with macrophages from one representative donor.
  • FIGS. 12A-12C demonstrate overcoming the HLA barrier.
  • FIG. 12A provides a schematic representation of the MHC class II and class I enhanceosomes. Targeting of CIITA, the master regulator of MHC class II expression, prevents MHC class II expression. The promoters of MHC class I genes are more complex, and thus deletion of NLRC5, a CIITA homologues regulating MHC class I expression, results in only a reduction of MHC class I expression.
  • FIG. 12A provides a schematic representation of the MHC class II and class I enhanceosomes.
  • CIITA the master regulator of MHC class II expression
  • the promoters of MHC class I genes are more complex, and thus deletion of NLRC5, a CIITA homologues regulating MHC class I expression, results in only a reduction of MHC class I expression.
  • FIG. 12B shows reduction of IFNg-induced MHC class I expression in NLRC5-/-CIITA-/ hPSCs.
  • WT, or the indicated KO HUES9 cells were stimulated with IFNg for 48hrs and subsequently stained for MHC class I expression, recorded by FACS. Deletion of the accessory chain B2M prevents MHC class I surface expression entirely, but will render these cells susceptible to NK cell killing.
  • FIG. 12C shows targeting strategy to selectively remove the polymorphic HLA genes HLA-A/B/C from the genome of hPSCs. Schematic representations of targeting strategy are provided. Also shown is PCR confirmation of the respective deletions in the genome of HUES 8.
  • FIGS. 13A-13E demonstrate knock-in (KI) strategy of tolerogenic factors into a safe harbor locus.
  • FIGS. 13A-13B provide schematic representation of the KI constructs.
  • FIG. 13C shows confirmation of the loss of HLA class I expression in two KI clones (C8 and C12).
  • FIG. 13D shows successful over expression of PD-L1 and CD47 in the HLA deficient KI clones C8 and C12 from the AAVS1 safe harbor locus.
  • FIG. 13E shows the ultimate goal is to reverse engineer the immunomodulatory activity of human trophoblasts (PD-L1, HLA-G, CD47 high) which induce tolerance to a semiallogeneic fetus (50% of paternal and thus foreign origin) during pregnancy.
  • PD-L1, HLA-G, CD47 high
  • FIGS. 14A-14B demonstrate functional immune-silent cells for
  • FIG. 14A shows confirmation of HLA expression in modified hPSC (HUES8). Loss of MHC class I expression was confirmed in two independent HLA- A/B/C -/-CIITA-/- KO clones - D1 and F2 - by FACS. Similar morphology of KO clone-derived endothelial cells (EC) was seen. IFNy-induced MHC class II expression in EC of the indicated genotypes, demonstrates loss of HLA class II in the HLA-A/B/C -/-CIITA-/- KO clones.
  • FIG. 14B shows a T cell proliferation assay (top panel) and a NK cell degranulation assay (bottom panel).
  • T cell proliferation assay (top panel), a CFSE-labelled T cell clone was used to assess T cell proliferation against EC derived from HUES9 of the indicated genotypes. Loss of CFSE signal is proportional to T cell proliferation, a proxy of the immunostimulatory activity of those cells. While WT EC trigger prominent T cell proliferation over a 7 day period,
  • T cell proliferation is reduced in the presence of two independent NLRC5-/-CIITA-/- KO clones and absent when co-incubated with B2M-/-CIITA-/- KO EC.
  • an HLA-deficient VSMC Dl, F2
  • PMA/Ionomycin or HLA-deficient 221 cells were used as positive control for NK degranulation.
  • NC negative control, NK cells only.
  • FIGS. 15A-15B demonstrate generation of preclinical data.
  • FIG. 15A shows improved engraftment of immune silent human pluripotent stem cells in humanized mice. NSG mice reconstituted with a human immune system (BLT), were
  • FIG. 15B shows introduction of an inducible Caspase9 (iCasp9) killing switch can ablate cells upon treatment with the CID dimerizer.
  • the inventions disclosed herein employ genome editing technologies (e.g., the CRISPR/Cas or TALEN systems) to reduce or eliminate expression of critical immune genes or, in certain instances, insert tolerance-inducing factors, in stem cells, rendering them and the differentiated cells prepared therefrom hypoimmunogenic and less prone to immune rejection by a subject into which such cells are transplanted.
  • genome editing technologies e.g., the CRISPR/Cas or TALEN systems
  • hypoimmunogenic generally means that such cell is less prone to immune rejection by a subject into which such cells are transplanted.
  • a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection by a subject into which such cells are transplanted.
  • genome editing technologies e.g., the CRISPR/Cas or TALEN systems
  • modulate e.g., reduce or eliminate
  • the inventions disclosed herein relate to a stem cell, the genome of which has been altered to reduce or delete critical components of HLA expression. Similarly, in certain embodiments, the inventions disclosed herein relate to a stem cell, the genome of which has been altered to insert one or more tolerance inducing factors.
  • the present invention contemplates altering target polynucleotide sequences in any manner which is available to the skilled artisan, for example, utilizing a TALEN, ZFN, or a CRISPR/Cas system.
  • CRISPR/Cas systems can employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;l(6)e60).
  • 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 type V system. It should be understood that although examples of methods utilizing CRISPR/Cas (e.g., Cas9 and Cpfl) and TALEN are described in detail herein, the invention is not limited to the use of these methods/systems. Other methods of targeting polynucleotide sequences to reduce or ablate expression in target cells known to the skilled artisan can be utilized herein.
  • the present inventions contemplate altering, e.g., modifying or cleaving, target polynucleotide sequences in a cell for any purpose, but particularly such that the expression or activity of the encoded product is reduced or eliminated.
  • the target polynucleotide sequence in a cell e.g., ES cells or iPSCs
  • a“mutant cell” generally refers to a cell with a resulting genotype that differs from its original genotype or the wild-type cell.
  • a“mutant cell” exhibits a mutant phenotype, for example when a normally functioning stem gene is altered using the CRISPR/Cas systems.
  • the target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell).
  • the target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).
  • the alteration is an indel.
  • “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof.
  • an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three.
  • the alteration is a point mutation.
  • “point mutation” refers to a substitution that replaces one of the nucleotides.
  • a CRISPR/Cas system can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence.
  • the alteration results in a knock out of the target polynucleotide sequence or a portion thereof.
  • knocking out a target polynucleotide sequence in a cell can be performed in vitro, in vivo or ex vivo for both therapeutic and research purposes.
  • Knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing those cells comprising the knocked out mutant allele into a subject).
  • knock out includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence or its expression product.
  • the alteration results in reduced expression of the target polynucleotide sequence.
  • the terms“decrease,”“reduced,”“reduction,” and “decrease” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt,“decreased,”“reduced,” “reduction,”“decrease” includes a decrease by at least 10% as compared to a reference level, for example a decrease 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 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • the terms“increased,”“increase” or“enhance” or“activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms“increased”,“increase” or“enhance” or“activate” means an increase of at least 10% as compared to a reference level, for example an increase of 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 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3- fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • “statistically significant” or“significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker.
  • the term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • the alteration is a homozygous alteration. In some embodiments, the alteration is a heterozygous alteration.
  • the alteration results in correction of the target polynucleotide sequence from an undesired sequence to a desired sequence.
  • CRISPR/Cas systems can be used to correct any type of mutation or error in a target polynucleotide sequence.
  • CRISPR/Cas systems can be used to insert a nucleotide sequence that is missing from a target polynucleotide sequence due to a deletion.
  • CRISPR/Cas systems can also be used to delete or excise a nucleotide sequence from a target polynucleotide sequence due to an insertion mutation.
  • CRISPR/Cas systems can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (e.g., to restore function to a target polynucleotide sequence that is impaired due to a loss of function mutation).
  • CRISPR/Cas systems can alter target polynucleotides with surprisingly high efficiency.
  • the efficiency of alteration is at least about 5%.
  • the efficiency of alteration is at least about 10%. In certain embodiments, the efficiency of alteration is from about 10% to about 80%. In certain embodiments, the efficiency of alteration is from about 30% to about 80%. In certain embodiments, the efficiency of alteration is from about 50% to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 85%. In some embodiments, the efficiency of alteration 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 efficiency of alteration is equal to about 100%.
  • 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.
  • CRISPR/Cas systems include a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one to two ribonucleic acids (e.g., gRNAs) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • CRISPR/Cas systems include a Cas protein or a nucleic acid sequence encoding the Cas protein and a single ribonucleic acid or at least one pair of ribonucleic acids (e.g., gRNAs) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • “protein” and“polypeptide” are used interchangeably to refer to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids) and include modified amino acids (e.g.,
  • polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants, and analogs of the above.
  • a Cas protein comprises one or more amino acid substitutions or modifications.
  • the one or more amino acid substitutions comprise a conservative amino acid substitution.
  • substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in a cell.
  • the Cas protein can comprise a peptide bond replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.).
  • the Cas protein can comprise a naturally occurring amino acid.
  • the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.).
  • a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).
  • a Cas protein comprises a core Cas protein.
  • Exemplary Cas core proteins include, but are not limited to Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9.
  • a Cas protein comprises a Cas protein of an E. coli subtype (also known as CASS2).
  • Exemplary Cas proteins of the E. Coli subtype include, but are not limited to Csel, Cse2, Cse3, Cse4, and Cas5e.
  • a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3).
  • Exemplary Cas proteins of the Ypest subtype include, but are not limited to Csyl, Csy2, Csy3, and Csy4.
  • a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4).
  • Exemplary Cas proteins of the Nmeni subtype include, but are not limited to Csnl and Csn2.
  • a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1).
  • Exemplary Cas proteins of the Dvulg subtype include Csdl,
  • a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7).
  • Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cstl, Cst2, Cas5t.
  • a Cas protein of the Tneap subtype include, but are not limited to, Cstl, Cst2, Cas5t.
  • Cas protein comprises a Cas protein of the Hmari subtype.
  • Exemplary Cas proteins of the Hmari subtype include, but are not limited to Cshl, Csh2, and Cas5h.
  • a Cas protein comprises a Cas protein of the Apem subtype (also known as CASS5).
  • Exemplary Cas proteins of the Apem subtype include, but are not limited to Csal, Csa2, Csa3, Csa4, Csa5, and Cas5a.
  • a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).
  • Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csml , Csm2, Csm3, Csm4, and Csm5.
  • a Cas protein comprises a RAMP module Cas protein.
  • Exemplary RAMP module Cas proteins include, but are not limited to, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.
  • the Cas protein is Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is Cas9 from any bacterial species or functional portion thereof.
  • Cas9 protein is a member of the type II CRISPR systems which typically include a trans-coded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas protein.
  • Cas 9 protein also known as CRISPR- associated endonuclease Cas9/Csnl
  • Cas 9 protein is a polypeptide comprising 1368 amino acids.
  • Cas 9 contains 2 endonuclease domains, including an RuvC-like domain (residues 7- 22, 759-766 and 982-989) which cleaves target DNA that is non-complementary to crRNA, and an HNH nuclease domain (residues 810-872) which cleave target DNA complementary to crRNA.
  • the Cas protein is Cpfl protein or a functional portion thereof. In some embodiments, the Cas protein is Cpfl from any bacterial species or functional portion thereof.
  • Cpfl protein is a member of the type V CRISPR systems.
  • Cpfl protein is a polypeptide comprising about 1300 amino acids.
  • Cpfl contains a RuvC-like endonuclease domain.
  • Cpfl cleaves target DNA in a staggered pattern using a single ribonuclease domain. The staggered DNA double- stranded break results in a 4 or 5-nt 5’ overhang.
  • “functional portion” refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence.
  • the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.
  • the functional portion comprises a combination of operably linked Cpfl protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex.
  • the present invention contemplates various ways of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas9).
  • exogenous Cas protein can be introduced into the cell in polypeptide form.
  • Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide.
  • “cell- penetrating polypeptide” and“cell-penetrating peptide” refers to a polypeptide or peptide, respectively, which facilitates the uptake of molecule into a cell.
  • the cell- penetrating polypeptides can contain a detectable label.
  • Cas proteins can be conjugated to or fused to a charged protein (e.g., that carries a positive, negative or overall neutral electric charge). Such linkage may be covalent.
  • the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate a cell (Cronican et al. ACS Chem Biol.2010;5(8):747-52).
  • the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into a cell.
  • PTDs protein transduction domain
  • Exemplary PTDs include Tat, oligoarginine, and penetratin.
  • the Cas protein comprises a Cas polypeptide fused to a cell -penetrating peptide.
  • the Cas protein comprises a Cas polypeptide fused to a PTD.
  • the Cas protein can 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 Cpfl).
  • the process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector.
  • the nucleic acid comprises DNA.
  • the nucleic acid comprises a modified DNA, as described herein.
  • the nucleic acid comprises mRNA.
  • the nucleic acid comprises a modified mRNA, as described herein (e.g., a synthetic, modified mRNA).
  • nucleic acids encoding Cas protein and nucleic acids encoding the at least one to two ribonucleic acids are introduced into a cell via viral transduction (e.g., lend viral transduction).
  • 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, as described herein (e.g., a synthetic, modified mRNA).
  • ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • at least one of the ribonucleic acids comprises tracrRNA.
  • at least one of the ribonucleic acids comprises CRISPR RNA (crRNA).
  • crRNA CRISPR RNA
  • a single ribonucleic acid comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • At least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • the ribonucleic acids of the present invention can be selected to hybridize to a variety of different target motifs, depending on the particular
  • the one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.
  • At least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7.
  • At least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7. In some embodiments, at least one ribonucleic acid comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-7.
  • each of the one to two ribonucleic acids comprises guide RNAs that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • one or two ribonucleic acids e.g., guide RNAs
  • one or two ribonucleic acids are complementary to and/or hybridize to sequences on the opposite strands of a target polynucleotide sequence.
  • the one or two ribonucleic acids are not complementary to and/or do not hybridize to sequences on the opposite strands of a target polynucleotide sequence.
  • the one or two ribonucleic acids are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence.
  • the one or two ribonucleic acids are complementary to and/or hybridize to offset target motifs of a target polynucleotide sequence.
  • the target motif is a 17 to 23 nucleotide DNA sequence. In some embodiments, the target motif is at least 20 nucleotides in length. In some embodiments, the target motif is a 20-nucleotide DNA sequence.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.
  • the target polynucleotide sequence in a cell is altered to reduce or eliminate expression and/or activity of one or more critical immune genes in the cell using a genetic editing system (e.g., TALENs, ZFN, CRISPR/Cas, etc.).
  • a genetic editing system e.g., TALENs, ZFN, CRISPR/Cas, etc.
  • the present disclosure provides that the target polynucleotide sequence in a cell is altered to delete a contiguous stretch of genomic DNA (e.g., delete one or more critical immune genes) from one or both alleles of the cell (e.g., using a CRISPR/Cas system).
  • the target polynucleotide sequence in a cell is altered to insert a genetic mutation in one or both alleles of the cell (e.g., using a CRISPR/Cas system).
  • the universal stem cells disclosed herein may be subject to complementary genome editing approaches (e.g., using a CRISPR/Cas system), whereby such stem cells are modified to both delete contiguous stretches of genomic DNA (e.g., critical immune genes) from one or both alleles of the cell, as well as to insert one or more tolerance-inducing factors, such as HLA-G, CD47, and/or PD-L1, into one or both alleles of the cells to locally suppress the immune system and improve transplant engraftment.
  • tolerance-inducing factors such as HLA-G, CD47, and/or PD-L1
  • the universal stem cells disclosed herein may be used, for example, to diagnose, monitor, treat and/or cure the presence or progression of a disease or condition in a subject (e.g., type 1 diabetes or multiple sclerosis).
  • a “subject” means a human or animal.
  • the subject is a human.
  • the subject is an adolescent.
  • the subject is treated in vivo, in vitro and/or in utero.
  • a subject in need of treatment in accordance with the methods disclosed herein has a condition or is suspected or at increased risk of developing such condition.
  • the universal stem cells are transplanted into a subject.
  • novel cells, compositions and methods that are useful for addressing such HLA-based immune rejection of transplanted cells.
  • the inventions disclosed herein relate to genomic modifications of one or more targeted polynucleotide sequences of the stem cell genome that regulates the expression of MHC-I and/or MHC-II human leukocyte antigens.
  • a genetic editing system is used to modify one or more targeted polynucleotide sequences.
  • a CRISPR/Cas system is used to delete the one or more targeted polynucleotide sequences and/or introduce indels into the one or more targeted polynucleotide sequences.
  • the efficient removal of the HLA barrier can be accomplished by targeting the polymorphic HLA alleles (HLA- A, -B, -C) directly and/or deletion of components of the MHC enhanceosomes, such as CIITA, that are critical for HLA expression.
  • HLA expression is interfered with.
  • HLA expression is interfered with by targeting individual HLAs (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).
  • multiple HLAs may be targeted at the same time.
  • HLA-B and HLA-C are adjacent and may be targeted simultaneously.
  • a 95 kb deletion of a stem cells genome using CRISPR/Cas may knock out HLA-B and HLA-C, as well as the promoters of the two genes.
  • a 13 kb deletion of a stem cells genome using CRISPR/Cas knocks out HLA- A, as well as the promoter of the gene.
  • the stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and/or HLA-DR) corresponding to MHC-I and/or MHC-II and are thus characterized as being hypoimmunogenic.
  • the stem cells disclosed herein have 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.
  • one or more of HLA-A, HLA-B and HLA-C may be“knocked-ouf’ of a cell.
  • a cell that has a knocked-out HLA-A gene, HLA-B gene, and/or HLA-C gene may exhibit reduced or eliminated expression of each knocked-out gene.
  • the stem cells disclosed herein have 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-II molecules: HLA-DP, HLA-DQ, and HLA-DR.
  • one or more indels are inserted into a transcriptional regulator of HLA class II expression (e.g., CIITA).
  • a cell that has indels inserted into CUT A (e.g., targeting exon 1) may exhibit reduced or eliminated expression of HLA- DP, HLA-DQ, and/or HLA-DR.
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the 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 cell or population thereof.
  • the contiguous stretch of genomic DNA can be deleted by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding the Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 1-2.
  • the present 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 acids direct Cas protein to and hybridize 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 the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1-2.
  • Cas clustered regularly interspaced short palindromic repeats-associated
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the 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 cell or population thereof.
  • the contiguous stretch of genomic DNA can be deleted by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding the Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 3-4.
  • the present 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 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 the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3-4.
  • Cas clustered regularly interspaced short palindromic repeats-associated
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the 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 cell or population thereof.
  • the contiguous stretch of genomic DNA can be deleted by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding the Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 5-6.
  • the present 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 acids direct Cas protein to and hybridize 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 the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 5-6.
  • Cas regularly interspaced short palindromic repeats-associated
  • the present disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof comprising a genome in which the 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 cell or population thereof.
  • the one or more indels can be introduced by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding the Cas protein and a ribonucleic acid consisting of SEQ ID NO: 7.
  • exon 1 of CIITA is targeted with the ribonucleic acid consisting of SEQ ID NO: 7 and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 1-2.
  • the present disclosure provides a method for introducing one or more indels in a cell comprising contacting the 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 Cas protein to and hybridizes to a target motif of the target CIITA polynucleotide sequence, wherein one or more indels are introduced into exon 1 of the CIITA polynucleotide sequence, and wherein the ribonucleic acid has a sequence of SEQ ID NO: 7.
  • exon 1 of CIITA is targeted with the ribonucleic acid consisting of SEQ ID NO: 7 and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 1-2.
  • one or more tolerogenic factors can be inserted or reinserted into genome-edited stem cell lines to create immune-privileged universal donor stem cells.
  • the universal stem cells disclosed herein have been further modified to express one or more tolerogenic factors.
  • Exemplary tolerogenic factors include, without limitation, one or more of HLA-G, PD-L1, and CD47. The expression of such tolerogenic factors may inhibit immune rejection.
  • a donor plasmid comprises a HLA-G expression cassette.
  • a donor plasmid comprises a PD-L1 expression cassette.
  • a donor plasmid comprises a CD47 expression cassette.
  • a donor plasmid comprises a PD-L1, HLA-G, and CD47 expression cassette.
  • a donor plasmid comprises a PD-L1 and CD47 expression cassette.
  • the donor plasmid comprising an expression cassette may target the AAVS1 locus of a stem cell (e.g., a hypoimmunogenic stem cell).
  • a stem cell e.g., a hypoimmunogenic stem cell
  • the donor plasmid targets the AAVS1 locus of a hypoimmunogenic stem cell with a ribonucleic acid, wherein the ribonucleic acid has a sequence of SEQ ID NO:
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the stem cell genome has been modified to express HLA-G.
  • the present disclosure provides a method for altering a stem cell genome to express HLA-G.
  • at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of HLA-G into a stem cell line.
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the stem cell genome has been modified to express PD-L1.
  • the present disclosure provides a method for altering a stem cell genome to express PD-L1.
  • at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of PD-L1 into a stem cell line.
  • the present disclosure provides a stem cell (e.g., a stem cell
  • hypoimmunogenic stem cell or population thereof comprising a genome in which the stem cell genome has been modified to express CD-47.
  • the present disclosure provides a method for altering a stem cell genome to express CD-47.
  • at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of CD-47 into a stem cell line.
  • the present disclosure provides a hypoimmunogenic stem cell (e.g., a stem cell modified to have ablated expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR) or population thereof comprising a genome in which the stem cell genome has been modified to express PD-L1, HLA-G, and CD47.
  • a hypoimmunogenic stem cell e.g., a stem cell modified to have ablated expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR
  • population thereof comprising a genome in which the stem cell genome has been modified to express PD-L1, HLA-G, and CD47.
  • the present disclosure provides a method for altering a stem cell genome to express PD-L1, HLA-G, and CD47.
  • the present disclosure provides a hypoimmunogenic stem cell (e.g., a stem cell modified to have ablated expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR) or population thereof comprising a genome in which the stem cell genome has been modified to express PD-L1 and CD47.
  • a method for altering a stem cell genome to express PD-L1 and CD47 is e.g., a stem cell modified to have ablated expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR
  • the inventions disclosed herein relate 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 expression of one or more tolerogenic factors.
  • the universal stem cells are HLA- A 7 , HLA-B 7 , HLA-C 7 , and ciITA mdel7mdel cells that exhibit increased expression of HLA-G, PD-L1, and CD47.
  • the stem cells exhibit one or more features.
  • the stem cells retain the differentiation potential, exhibit reduced T cell response, exhibit protection from NK cell response, and exhibit reduced macrophage engulfment.
  • the universal stem cells may retain pluripotency, perform tri-lineage differentiation, and retain normal karyotype.
  • the universal stem cells may retain expression of one or more of NANOG, OCT4, SSEA3, and TRA-1-60.
  • the universal stem cells are differentiated into the three germ layers (e.g., ectoderm, mesoderm, and endoderm) and maintain expression of all lineage markers.
  • the universal stem cells demonstrate reduced T cell-mediated adaptive immune responses.
  • T cells e.g., CD4 + and CD8 + T cells
  • T cells exhibit reduced priming and activation against the universal stem cells.
  • T cells exhibit reduced cytokine secretion against the universal stem cells.
  • the reduced expression of HLA-I and HLA-II molecules may result in reduced CD4 + and CD8 + T cell priming against the universal cells.
  • the expression of PD-L1 further suppresses activation of CD8 + T cells.
  • the universal stem cells are protected from NK cell- mediated rejection.
  • the universal stem cells may be protected from NK cell-mediated rejection as a result of HLA-G expression.
  • the universal stem cells exhibit reduced macrophage engulfment. Overexpression of CD47 and/or expression of PD-L1 in the universal cells may minimize or inhibit macrophage engulfment of the universal cells.
  • the term“consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional
  • compositions, methods, kits and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • ablating HLA class I is necessary to prevent the presentation of cellular peptides to cytotoxic CD8 + T cells, given that HLA class I molecules are expressed in virtually all nucleated cells.
  • ablation of HLA class II needs to be considered, since they are also highly polymorphic and can be present in certain hPSC-derived donor cell types, in particular in professional antigen presenting cells (APCs) and endothelial cells (ECs) upon IFNy stimulation (Ting and Trowsdale, 2002).
  • CRISPR/Cas9 genome-editing system provided a tool to interfere with HLA class I expression in hPSCs or hematopoietic cells by knocking out the accessory chain beta-2-microglobulin (B2M) (Mandal et ak, 2014; Mattapally et ak, 2018; Meissner et ak, 2014; Riolobos et ak, 2013; Wang et ak, 2015), and to eliminate HLA class II expression by targeting its transcriptional master regulator, CIITA (Chen et ak, 2015; Mattapally et ak, 2018).
  • B2M beta-2-microglobulin
  • B2M also prevents the surface expression of nonpolymorphic nonclassical HLA class lb molecules HLA-E and HLA-G, which are required to maintain NK cell tolerance (Ferreira et ak, 2017; Lee et ak, 1998b). Moreover, it has been found that B2M- deficient cells are still rejected by allogeneic CD8+ T cells (Glas et ak, 1992).
  • individual deletion of the HLA-A/-B/-C genes may represent a more favorable strategy to protect the donor cells from CD8 + T cell-mediated cytotoxicity without losing HLA class lb protective function.
  • TCR HLA-T cell receptor
  • ectopic expression of the T cell checkpoint inhibitors PD-L1 and CTLA-4Ig has been shown to protect stem cells from rejection in a humanized mouse model (Rong et ak, 2014). Yet, this approach left the HLA- barrier intact, which may result in hyperacute rejection of the engrafted cells precipitated by preexisting anti-HLA antibodies (Iniotaki-Theodoraki, 2001; Masson et ak, 2007).
  • CTLA-4Ig can also impair T regulatory cell (Treg) homeostasis and function, possibly jeopardizing the establishment of operational immune tolerance (Bour-Jordan et ak, 2004; Salomon and Bluestone, 2001).
  • Innate immune cells such as NK cells and macrophages, serve an important role in priming adaptive immune responses in many contexts, including chronic graft rejection.
  • a major concern associated with B2M deletion is that this strategy renders the donor cells vulnerable to NK cell mediated killing due to“missing self’ (Raulet, 2006).
  • Gornalusse et ak expressed a B2M-HLA-E fusion construct in B2M- deficient cells to overcome NK cell-mediated lysis (Gornalusse et ak, 2017).
  • HLA-G an NK cell inhibitory ligand expressed at the maternal-fetal interface during pregnancy, that acts through multiple inhibitory receptors (Ferreira et al., 2017; Pazmany et al., 1996), might be a better candidate to fully overcome NK cell responses.
  • macrophages which contribute to rejection of transplanted cells, may be controlled by expression of CD47, a“don’t-eat- me” signal that prevents cells from being engulfed by macrophages (Chhabra et al., 2016; Jaiswal et al., 2009; Majeti et al., 2009).
  • this approach has not yet been explored to protect hPSCs and their differentiated derivatives from macrophage engulfment.
  • a convincing strategy to target both adaptive and innate immunity is yet to be proposed.
  • the CRISPR/Cas9 system can be used to selectively excise the genes encoding the polymorphic HFA class I members, HFA- A/-B/-C, from the genome of hPSCs. Moreover, its multiplexing capacity allows for the simultaneous ablation of HFA class II gene expression using a single guide RNA targeting CIITA.
  • the resulting polymorphic HFA-deficient,“immune-opaque” cells were further modified to express the immunomodulatory factors PD-F1, HFA-G and CD47, which target immune surveillance by T cells, NK cells, and macrophages, respectively, further muting alloresponses in vitro and in vivo. Combining these and other genetic modifications may ultimately result in universal“off-the-shelf’ cell products suitable for transplantation into any patient.
  • HFA- A, HFA-B, and HFA-C are highly homologous
  • designing specific short guide RNAs (sgRNAs) targeting the coding regions of each gene using the CRISPR/Cas9 genome-editing system proved challenging.
  • sgRNAs short guide RNAs
  • a dual guide multiplex strategy was employed targeting non coding regions adjacent to these genes to simultaneously excise all three from the genome of an hPSC line (HUES8).
  • HFA-B and HFA-C are adjacent, whereas HFA- A is located nearer the telomere.
  • sgRNAs were designed at each site, upstream of HLA-B and downstream of HLA-C (FIG. 1 A).
  • the predicted 95 kb deletion also includes the promoters of the two genes, defined as H3K27 Ac-positive areas on the UCSC Genome Browser.
  • one sgRNA was designed upstream and another sgRNA downstream of HLA-A (FIG.
  • the predicted 13 kb deletion includes the HLA-A promoter, according to the UCSC Genome Browser. Both deletions were confirmed by PCR amplicons spanning the predicted Cas9 cutting sites (FIGS. 6A-6B). Ablation of HLA -A/- B/-C proteins in the final HLA knock-out clone (KO), was verified by flow cytometry (FIG. 1C).
  • Targeting CIITA the master regulator of HLA class II expression, is a well- documented strategy to collectively ablate the expression of the three highly polymorphic HLA class II alleles, HLADP/-DQ/-DR (Krawczyk and Reith, 2006; Reith and Mach, 2001).
  • a sgRNA targeting exon 1 of CIITA with high cutting efficiency was previously reported (FIG. ID) (Ding et ak, 2013). This sgRNA was used in combination with the sgRNAs targeting the 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 cutting site.
  • PCR amplicons were Sanger sequenced to identify biallelic frame shifts (FIGS. 6C-6D).
  • ECs endothelial cells
  • 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 unaffected by genome editing (FIG. 6E).
  • induction of HLA-DR expression upon IFNy stimulation was abolished in KO ECs (FIG. IE).
  • the KO hPSC clone with a genotype of HLA-A-/-HLA-B-/-HLAC-/-CIITAindel/indel was generated following the workflow depicted in FIG. 6F.
  • the results demonstrate that multiplex CRISPR/Cas9 genome editing allows for combined and highly specific ablation of polymorphic HLA class I and II gene expression in hPSCs.
  • HLA knockout cells would likely still be susceptible to innate immune cells involved in an alloresponse, such as NK cells and macrophages, prompting the exploration of the effect of introducing immunomodulatory factors based on the following rationale: 1) while the non-polymorphic HLA-E gene will be left intact, its surface expression will likely be severely impaired by the removal of polymorphic HLA class I genes, as the predominant peptides presented by HLA-E are leader peptides derived from other class I molecules (Braud et al., 1998b).
  • HLA-G HLA knockout cells
  • Macrophages are attracted by cytokines secreted at the site of engraftment and are primed to phagocytose foreign cells by antibody binding.
  • CD47 which binds to signal regulatory protein alpha (SIRPa) on the surface of macrophages, acting as a“don’t eat me” signal, is significantly increased in certain types of tumors and helps them escape macrophage engulfment (Betancur et ak, 2017; Jaiswal et ak, 2009; Willingham et ah, 2012; Zhao et ak, 2016). Therefore, it was aimed to overexpress CD47 in HLA knockout cells. 3) HLA-G can present classical peptides derived from intracellular proteins to T cells (Diehl et ak, 1996), which would potentially re-expose the cell lines to CD8+ T cell immune surveillance.
  • SIRPa signal regulatory protein alpha
  • Lurthermore gdT cells can directly recognize antigens and initiate a cytotoxic response (Vantourout and Hayday, 2013).
  • PD-L1 a T cell checkpoint inhibitor that engages the PD-1 receptor on activated T cells, directly suppressing T cell activities (Riley, 2009).
  • PD-L1 expression may also contribute to protecting transplanted cells from innate immune rejection by inhibiting PD-1+ NK cells (Beldi-Lerchiou et ak, 2016; Della Chiesa et ak, 2016) and PD-1+ macrophages (Gordon et ak, 2017).
  • CD47 expression cassette both driven by a CAGGS promoter flanked by arms homologous to the AAVS1 locus (LIG. 1G).
  • the donor plasmids were electroporated together with a sgRNA targeting the AAVS1 locus into the HLA-A-/-HLAB-/-HLA-C- /-CIITAindel/indel clone. Integration of the expression cassettes into the AAVS1 locus was verified by PCR (FIG. 6G).
  • Two clones were isolated following the workflow in FIG. 6F and analyzed by flow cytometry; one named KI-PHC that expressed PD-L1, HLA-G, but did not significantly overexpress CD47, compared to WT cells (FIGS.
  • FIG. 1H-1I 1H-1I
  • FIG. 1J and FIG. 7A a second one named KI-PC that expressed PD-L1 and displayed elevated CD47 level
  • FIG. 7B Surface HLA-A2 levels were checked by flow cytometry in both KI clones and confirmed HLA class la ablation (FIG. 7B).
  • KI-PHC and KI-PC hPSCs were differentiated into CD 144+ ECs (FIG. 7C), and no HLA-DR expression was observed by flow cytometry following IFNy stimulation (FIG. 7D).
  • immunomodulatory factors were successfully inserted into the AAVS1 safe harbor locus of HLA class la and II null cells.
  • three engineered hPSC lines: KO, KI-PHC, and KI-PC (FIG. IF) were generated.
  • the engineered hPSCs were differentiated into vascular smooth muscle cells (VSMCs).
  • WT, KO, KI-PHC and KI-PC VSMCs expressed equivalent levels of the VSMC marker CD140b (PDGFRB), confirming similar differentiation efficiencies (FIG. 7E).
  • PDGFRB vascular smooth muscle cells
  • FIG. 7F a subpopulation with modestly higher expression of PD-L1 and HLA-G was observed, compared to WT VSMCs, and a major population displaying significantly elevated levels of PD-L1 and HLA-G (FIG. 7F).
  • FIG. 7F increased CD47 expression in KI-PHC VSMCs was not observed (FIG. 7F), which could be a result of incomplete expression from the targeting cassette, where all three gene products are linked by a 2A-peptide (FIG. 1G).
  • FIG. 7F a small subpopulation with modestly higher, and a major population with highly elevated levels of PD-L1 and CD47 in KI-PC VSMCs was observed, compared to WT VSMCs (FIG. 7F).
  • HLA-E protein levels on the cell surface were greatly reduced in KO VSMCs (FIG. 7G), which was not due to an impaired HLA-E gene expression in KO VSMCs (FIG. 7H).
  • surface HLA-E expression of KI-PHC VSMCs was not restored by HLA-G expression (FIG. 7G).
  • HLA-G surface trafficking was unimpaired in the KI-PHC VSMCs (FIG. 7F), providing further incentive to introduce this tolerogenic factor into the engineered cell products to compensate for the reduction of HLA-E surface expression in an HLA-A/- B/-C null background.
  • the 21 top ranked in silico predicted exonic off-target sites were PCR amplified from the engineered hPSC lines as well as from the parental WT hPSCs.
  • Sanger sequencing of the PCR products did not reveal any unwanted edits on these sites except for the pseudogene HFA-H (HFE), which displayed a perfect match to the sgRNA upstream of HEA-A used to delete HLA-A from the genome (FIG. 2D and FIG. 8). More extensively, target capture sequencing was performed for all of the 648 predicted off-target sites for the eight sgRNAs used in this study.
  • HFE pseudogene HFA-H
  • HFA-H HFE was confirmed as an off-target in all three cell lines.
  • an intronic off-target site was detected in TRAF3 in all three cell lines resulting from targeting HFA-C, as well as an intronic off-target site in CPNE5 in the KI-PC cell line as a result of the AAVS1 sgRNA (FIG. 2E and FIG. 9).
  • the engineered hPSC lines retained pluripotency and their capacity to differentiate into cells of all three germ layers, as well as into VSMCs and ECs with similar differentiation efficiencies to their WT counterparts.
  • T cell proliferation T cell proliferation, activation, cytokine secretion, and killing assays.
  • HLA I expression is modest in hPSCs (de Almeida et ah, 2013; Drukker et ak, 2002), the engineered as well as WT hPSCs were differentiated into ECs, which express both HLA I and II following IENg stimulation, or into VSMCs, which only express HLA I, before being used in the respective immunoassays.
  • T cell proliferation assays WT, KO, and KI-PHC ECs were pre-treated with IENg for 48 hours and subsequently co-cultured with CFSE-labeled allogeneic CD3+ T cells for five days. T cells were then stained for CD3/4/8 and analyzed for dilution of the CFSE signal by flow cytometry as a read-out for T cell proliferation in the different T cell subpopulations (FIG. 10A). FACS plots of one representative T cell donor are shown in FIG. 10B.
  • CD3+ T cells As predicted, the percentage of total proliferating T cells (CD3+) was reduced when incubated with KO ECs (4.17% ⁇ 0.89% SEM) or KI-PHC ECs (3.87 % ⁇ 0.73% SEM), compared to WT ECs (8.29% ⁇ 1.23% SEM) (FIG. 3 A, left panel).
  • CD4+ T cells followed a similar pattern, with WT ECs (5.03%
  • CD 8+ cytotoxic T cells exhibited significantly reduced proliferation when co-cultured with KO ECs (7.71% ⁇ 1.89% SEM) or KI-PHC ECs (5.95% ⁇ 1.48% SEM), as compared to WT ECs (14.32% ⁇ 2.39% SEM) (FIG. 3A, right panel).
  • KO ECs 7.71% ⁇ 1.89% SEM
  • KI-PHC ECs 5.95% ⁇ 1.48% SEM
  • WT ECs (14.32% ⁇ 2.39% SEM
  • FIG. 3B Utilizing the same co-culture of T cells with ECs as target cells, the expression of the T cell activation markers CD25 and CD69 was examined (FIG. 3B). Reduced percentages were found of CD25+ and CD69+ T cells (CD3+) in co-cultures with KI- PHC ECs (4.91% ⁇ 0.74% SEM; 5.04% ⁇ 1.24% SEM) or KO ECs (5.12% ⁇ 0.77% SEM; 5.40% ⁇ 1.29% SEM), when compared to T cells co-incubated with WT ECs (6.43% ⁇ 0.71% SEM; 9.30% ⁇ 1.51% SEM) (FIG. 3B). The same trends were observed in the CD4+ and the CD8+ cell populations (FIG.
  • LDH lactate dehydrogenase
  • VSMCs was measured as a surrogate for T cell cytotoxicity. In this setting, only the CD8+ T cells were expected to be activated by HLA I-TCR engagement, given that VSMCs solely express HLA I. It was found that the CD8+ T cell cytotoxicity against KI-PHC VSMCs (15.31% ⁇ 4.52% SEM) was the lowest when compared to KO (18.86% ⁇ 4.34% SEM) and WT (37.65% ⁇ 7.64% SEM) VSMCs (FIG. 3D). This observation suggests that the CD8+ T cell cytotoxicity was suppressed even further by PD-L1 in KI-PHC VSMCs, consistent with the results of the CD8+ T cell
  • WT teratomas displayed a slower increase in volume compared to KO teratomas seven days after injection of CD8+ T cells, which was not due to a slower growth rate of the WT teratomas themselves (FIGS. 4B-4C). These results suggest that the KO teratomas were protected against T cell-mediated rejection. Moreover, although not significant, the average volumes of the KI-PHC and KI-PC teratomas were also larger than that of the WT teratomas 7 days post T cell infusion (FIG. 4B).
  • teratomas derived from both, the KO and KI cell lines displayed reduced T cell infiltration, as evidenced by qPCR for the human effector T cell markers CD 8 and IL- 2 (FIG. 4D), as well as by histology (FIG. 4E).
  • FIG. 4D qPCR for the human effector T cell markers CD 8 and IL- 2
  • FIG. 4E histology
  • NK cell activation Due to the lack of HLA la molecules and impaired HLA-E surface expression, the KO hPSCs and their derivatives were expected to be vulnerable to NK cell- mediated lysis, whereas the KI-PHC cell line should be protected from NK cell- mediated rejection as a result of HLA-G expression.
  • allogenic NK cells were isolated from healthy donors and co-incubated with WT, KO, or KI- PHC VSMCs. CD56+ NK cells were analyzed by flow cytometry for surface expression of the degranulation marker CD107a as a readout of NK cell activation (FIG. 1 IB).
  • NK cell degranulation in the presence of KO VSMCs was not significantly higher than with WT VSMCs (10.16% ⁇ 2.96% SEM) (FIG. 5A), suggesting the lack of an NK cell activation signal on hPSC-derived VSMCs.
  • FIG. llC FACS plots of one representative donor are shown in FIG. llC.
  • the FDH released from apoptotic VSMCs after coincubation with NK cells was also examined to quantify NK cell cytotoxicity. Consistent with NK cell degranulation, it was observed that NK cell cytotoxicity was reduced when NK cells were incubated with KI-PHC VMSCs (FIG. 5B).
  • VSMCs (FIG. 7F)
  • VSMC differentiated from the KI-PC cell line was used in these assays, which displayed much higher CD47 level than WT VSMC (FIG. 7F).
  • a CD47 knockout (CD47-/-) cell line was generated as a positive control for macrophage engulfment and verified the loss of CD47 cell surface expression by flow cytometry (FIG. 11D).
  • pHrodo-Red labelled VSMCs differentiated from WT, CD47- /- and KI-PC cells were either treated with staurosporine (STS) to induce apoptosis or left untreated and then incubated with isolated allogeneic macrophages from healthy donors.
  • STS staurosporine
  • the polymorphic HLA class la genes, HLA-A/-B/-C were specifically excised, while leaving the genes B2M and the nonpolymorphic HLA class lb genes HLA-E, -L and -G intact. While the resulting 95 kb deletion contains not only HLA-B/-C genes, but also MIR6891 and four pseudogenes, there were no observed changes in growth rate or differentiation efficiency in the KO or KI cell lines.
  • HLA-E surface expression was not restored by the expression of HLA-G in KI-PHC cells, which was inconsistent with a previous report that the leader peptide from HLA-G is sufficient to promote HLA-E surface trafficking (Lee et ak, 1998a).
  • the HLA knockout (KO) hPSC line was generated by genome editing using seven different sgRNAs, and KI-PHC and KI-PC hPSC were clones derived from the KO line and edited by an additional sgRNA targeting the AAVS1 locus.
  • KI-PHC and KI-PC hPSC were clones derived from the KO line and edited by an additional sgRNA targeting the AAVS1 locus.
  • Out of 648 predicted off-target sites for the eight sgRNAs used only one exonic off-target event was observed in the transcribed pseudogene HLA-H, as a result of one of the sgRNAs used to delete HLA-A from the genome. If translated, the observed 2 bp deletion found in both alleles would result in a frameshift-causing mutation.
  • HLA-H HLA-H
  • RNP sgRNA/Cas9 ribonucleoprotein complexes
  • NK cells While acute graft rejection is mainly T cell-mediated, the role of other immune cells such as macrophages, NK cells, and B cells must also be considered with regards to engraftment and long-term survival of therapeutic cells.
  • the NK cell assays suggest that HLA-G expression was able to control NK cell activities.
  • PD-L1 which was co-expressed in both KI cell lines, can also impact the activities 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 phenotypes.
  • ADCC antibody- dependent cellular cytotoxicity
  • T1D type 1 diabetes
  • multiple sclerosis where one particular cell type is attacked by the patient’s own immune system and needs replacement.
  • HLA-A upstream 5’-GCCGCCTCCCACTTGCGCT-3’ (SEQ ID NO: 1)
  • HLA-B upstream_2 5’ -TCCCTAAATATGGTGTCCCT-3’ (SEQ ID NO: 4)
  • HLA-C downstream_l 5’ -GTG ATCCGGGT ATGGGC AGT-3 (SEQ ID NO: 5) HLA-C downstream_2: 5’ -TGATCCGGGTATGGGCAGTG-3’ (SEQ ID NO: 6) CUT A: 5’ -TCCATCTGGTCATAGAAG-3’ (SEQ ID NO: 7)
  • gRNA_AAVSl-T2 5’ -GGGGCCACTAGGGACAGGAT-3’ (SEQ ID NO: 8) PCR and qPCR Probes/Primers
  • Green_F 5’ -CACTCAGAGCAAAGGTCAGATG-3’ (SEQ ID NO: 13)
  • 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)
  • 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)
  • CTBS_F 5’ -GCCCTCATCGAGTGGTC AAA-3’ (SEQ ID NO: 31)
  • CTBS_R 5’ -CCGCTAGACCTGCTGCTATG-3’ (SEQ ID NO: 32)
  • AFP_F 5’ - AAATGCGTTTCTCGTTGCTT-3’ (SEQ ID NO: 63)
  • BRACHYURY_F 5’ - AATTGGTCCAGCCTTGGAAT-3’ (SEQ ID NO: 67)
  • BRACHYURY_R 5’-CGTTGCTCACAGACCACA-3’ (SEQ ID NO: 68)
  • FLK1_R 5 -C ACG ACTCC ATGTTGGTC AC-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)
  • HLA-E Hs03045171_ml
  • CD8 Hs00233520_ml
  • Isotype 1 Mouse IgG2b, k Isotype Control (APC-conjugated), Biolegend,
  • Isotype 2 Mouse IgG2b, k Isotype Control (PE-conjugated), Biolegend, Cat#401208
  • Isotype 3 Mouse IgG2a, k Isotype Control (PE-conjugated), Biolegend, Cat#400214
  • HUES8 cells (Cowan et ak, 2004) were grown on Geltrex (Life Technologies) pre-coated plates and cultured in mTeSRl (StemCell Technologies) supplemented with penicillin/streptomycin. For passaging, cells were dissociated with Gentle Cell Dissociation Reagent (StemCell Technologies) for 5-10 min and replated in fresh media supplemented with RevitaCellTM (ThermoFisher Scientific). For
  • HUES8 cells were dissociated into singles cells and 10 million cells were electroporated with 50 pg of pCas9_GFP (Addgene #44719) and a total of 50 pg of gRNA plasmid for gene knockout.
  • pCas9_GFP Allergan, Inc.
  • gRNA_AAVSl-T2 Allergan, Inc.
  • 40 pg of double-stranded donor plasmid were collected 48 hrs post-electroporation.
  • GFP-expressing cells were enriched by FACS (FACS Aria II, BD Biosciences) and replated on 10 cm tissue-culture plates at 15,000 cells/plate in fresh media supplemented with RevitaCellTM, to allow single cell colony formation.
  • FACS Fluorescence Activated Cell Sorting
  • 48 hrs post-electroporation cells were selected by blasticidin (ThermoFisher Scientific) at 2 pg/ml for 5 days. Cell colonies were then manually picked and expanded.
  • sgRNAs Five hundred base pairs of each region upstream or downstream of HLA- A/B/C were amplified from HUES8 or HEK293T cells and Sanger-sequenced (Genewiz). The sequence conserved between the two cell lines was chosen as reference sequence, and sgRNAs were designed using the CRISPR design tool developed by Feng Zhang’s lab at MIT (available at: crispr.mit.edu) and CCTop (Stemmer et ah, 2015). Top ranked sgRNAs were picked and cloned into a gRNA expression vector (Addgene #41824).
  • the gRNA plasmid was then transfected into HEK293T cells, genomic DNA was extracted and PCR amplicons covering the cutting site were analyzed by TIDE (available at: tide.nki.nl) for on-target efficiency. Single guide RNAs with the highest on-target activities were used for genome editing in HUES8 cells.
  • TIDE available at: tide.nki.nl
  • Single guide RNAs with the highest on-target activities were used for genome editing in HUES8 cells.
  • the ORFs of PD-L1, HLA-G, and CD47 were individually cloned and connected by 2A sequence using Gibson
  • HLA-B/-C 7 efficiency 1.56%
  • HLA-B/-C +/_ efficiency 7.8%
  • the homozygous clones were further verified for ablation of HLA-B/-C mRNA expression by RT-PCR and normal karyotypes were confirmed by nCounter Human Karyotype Assay (data not shown).
  • HFA-A/CIITA sgRNAs was applied to one karyotypically normal heterozygous clone, and the same screening strategies were employed.
  • one homozygous clone HLA-A /_ CIITA mdel/mdel
  • FACS analysis revealed that this clone was an admixed clone, which still retained 1% HFA-A + cells.
  • a pure homozygous clone HFA Knockout, KO was obtained.
  • EC Human endothelial cells
  • VSMC vascular smooth muscle cells
  • CHIR99021 (Cayman Chemical) and 25 ng/ml BMP4 (Peprotech) for 3 days to induce lateral mesoderm. Media were then replaced with StemPro-34 supplemented with 200 ng/ml VEGF (Peprotech) and 2 mM forskolin (Abeam) for 2 days to induce EC. Cells were then enriched for CD144 + cells using MACS cell separation (Miltenyi Biotec). The CD144 + cells were plated on Fibronectin (Corning)-coated plates in EBMTM-2 supplemented with EGMTM-2 BulletKitTM (Lonza) for further
  • VSMC differentiation for at least 7 days.
  • ESCs were plated in the same media for 3 days as for EC differentiation.
  • media were changed to N2B27 supplemented with 12.5 ng/ml PDGF-BB (Peprotech) and 12.5 ng/ml
  • Isolated T cells were cultured in X-VIVO 10 (Lonza) media supplemented with 5% Human AB Serum (Valley Biomedical), 5% Fetal Bovine Seram, 1 % Penicillin/Streptomycin, GlutaMAX, MEM Non-Essential Amino Acids (ThermoFisher Scientific), and 20 U/ml IL-2 (Peprotech).
  • Isolated NK cells were cultured in RPMI 1640 with L-Glutamine (Corning) supplemented with 10% Fetal Bovine Seram and 1% Penicillin/Streptomycin.
  • Isolated monocytes were differentiated into macrophages in RPMI 1640 supplemented with 10% Fetal Bovine Serum, 1% Penicillin/Streptomycin, and 25-50 ng/ml M-CSF (Peprotech). Flow Cytometry
  • FBS containing 1% Fetal Bovine Seram was used as washing and staining buffer; PBS containing 4% FBS was used as blocking buffer.
  • FcR blocking reagent Miltenyi Biotec
  • immune cells or other dissociated single cells were washed once and blocked with blocking buffer on ice for 20 min. Cells were stained with antibodies on ice for 30-60 min and washed twice before analysis on a FACSCaliburTM or FSR II (BD Biosciences). The data were plotted using FlowJo software (BD).
  • VSMCs When VSMCs were used, cells were first treated with mitomycin (Fisher Scientific). One hundred thousand ECs or VSMCs were plated on 24-well plates and treated with IFNy (100 ng/ml) for 48 hrs before the assay. On day 0 of co-incubation, isolated CD3+ T cells were labeled with CellTraceTM CFSE (ThermoFisher Scientific) following the manufacturer’s instructions. Adherent ECs or VSMCs were washed twice with PBS before co-incubation with 500k CFSE-labeled T cells in T cell culture media supplemented with 20 U/ml IE-2 for 5 days.
  • mitomycin Fisher Scientific
  • T cells were then stained with anti-CD3/4/8 antibodies before being analyzed on an LSR II for CFSE intensity.
  • T cells cultured for 5 days without target cells were used as negative control.
  • T cells treated with DynabeadsTM Human T- Activator CD3/CD28 beads (ThermoFisher Scientific) for 5 days served as positive control.
  • ESC-derived ECs were used as target cells.
  • the conditions for co-culture were the same as in the T cell proliferation assay, except that the T cells were not labeled.
  • T cells were stained for T cell activation markers before being analyzed on an LSR II.
  • supernatants were collected and analyzed by customized MSD U-PLEX Platform (Meso Scale Discovery) following manufacturer’s instructions.
  • T cells or target cells cultured for 5 days were used as negative control.
  • ESC-derived VSMCs were used as target cells.
  • T cell killing assay the conditions for co-incubation were the same as in the T cell activation assay.
  • NK cell killing assay 40K VSMCs and NK cells at the indicated effector/target ratios were co-incubated in 200 m ⁇ NK cell medium in 96-well U bottom for 20 hrs before the supernatants were harvested. After co-incubation, supernatants were collected and analyzed by PierceTM FDH Cytotoxicity Assay Kit (ThermoFisher Scientific) following the manufacturer’s instructions.
  • T cell medium or NK cell medium RPMI- 10.
  • T/NK cells cultured alone or target cells cultured alone were used as controls for spontaneous FDH release.
  • Fysed target cells at endpoint were used as maximum FDH release.
  • HUES8-derived embryoid bodies as previously described (Gomalusse et ak, 2017). Briefly, the embryoid bodies were induced in suspension for 5 days followed by attachment culture for another 4 days. CD8 + T cells were then co-cultured with attached embryoid body cells for pre-sensitization. Extracellular matrix from xenogeneic resources such as Gelatin was avoided during this process to prevent unspecific T cell activation.
  • mice Male immunodeficient SCID Beige mice (Taconic) aged 8-10 weeks were used for teratoma formation. Two million HUES 8 cells were encapsulated in a blood clot, and the blood clot was inserted subcutaneously into each flank of the SCID Beige mice. Teratoma size was measured by caliper weekly after the teratoma became palpable. Four to six weeks after hESC transplantation, one million pre-sensitized allogeneic human CD8 + T cells were injected via tail vein into the mice.
  • teratoma size was measured on day 2, day 5, and day 7; teratoma size was also measured 2 days before the T cell injection.
  • the teratoma were harvested and analyzed by qPCR and hematoxylin and eosin (H&E) staining.
  • H&E hematoxylin and eosin
  • VSMCs Three hundred thousand adherent ESC-derived VSMCs were seeded in 24- well plates 24 hrs before the assay. The next day, VSMCs were washed once with PBS before co-incubation with 100K freshly isolated NK cells in NK cell media supplemented with a-CD107a APC (Biolegend) and eBioscienceTM Protein Transport Inhibitor Cocktail (ThermoFisher Scientific). After NK cells were added into the wells, the plate was spun down at 2,000 rpm for 5 min to achieve sufficient effector- target contact. After a 20 h-co-incubation the NK cells were stained with a-CD56 PE (Biolegend) before analysis on a FACSCaliburTM for CD107a cell surface expression.
  • a-CD56 PE Biolegend
  • NK cell cultures without target cells were used as negative control.
  • NK cells treated with Cell Activation Cocktail (without Brefeldin A), which includes PM A (phorbol 12-myristate- 13-acetate) and ionomycin, were used as positive control for degranulation.
  • Monocytes were isolated from donor blood via negative selection using RosetteSepTM Human Monocyte Enrichment Cocktail (StemCell Technologies). Monocytes were plated in serum- free medium for adhesion and maturation into macrophages for one to three weeks in RPMI 1640 supplemented with 10% FBS, 1% Penicillin/Streptomycin, and 25 ng/ml of M-CSF (Peprotech). Macrophages were replated in 96-well m-plates (ibidi) at a density of 100K/well two days before the assay. For the assay, differentiated VSMCs were pretreated with 200 nM
  • VSMCs were dissociated and labeled with pHrodo-Red (IncuCyte) for 1 h in 37 °C. Thirty thousand labeled VSMCs were added into each well containing macrophages, and the co-incubated culture was immediately transferred into the Celldiscover 7 live cell imaging platform (Zeiss). One image per well of the red fluorescence emission upon phagocytic engulfment was acquired every 20 min for 6 hrs. Total integrated intensity (mean fluorescence intensity*total area) was analyzed for each image using the ZEN imaging software (Zeiss). The pHrodo-Red + particles indicate phagosomes within the macrophages that have engulfed VSMCs.
  • the following four CRISPR sgRNAs were used to target the first coding exon of CD47 in HUES 8 cells.
  • a doxycycline-inducible lentiviral Gateway vector (Invitrogen) containing the PD-L1 ORF was constructed by PCR amplification.
  • PD-L1 expression lentiviruses were packaged by transfecting HEK293T cells with the PD-L1 -expressing vector and the packaging plasmids pMDL, pVSVG, and pREV.
  • Medium containing lentiviral particles was collected 48 hr post-transfection and used to transduce VSMCs along with lentiviral particles encoding the doxycycline-binding transactivator rtTA.
  • VSMCs were treated with doxycycline (10 pg/ml) to induce PD-L1 expression that was verified by FACS.
  • Assessment of T cell proliferation against VSMCs overexpressing PD-L1 was performed as described above, except for a 7-day-co- incubation and the presence of doxycycline throughout the co-incubation. No effect on T cell proliferation was observed by the addition of doxycycline.
  • PBS containing 0.05% Tween-20 was the washing buffer between each step after cells were fixed. Briefly, cells were washed with PBS, fixed with 4%
  • Triton X-100 0.1% Triton X-100.
  • Cells were blocked with 4% Donkey Serum (Jackson ImmunoResearch Laboratories) at 4 °C overnight and incubated with appropriate primary antibodies diluted in blocking buffer at RT for 1 hr. Cells were then incubated with Alexa Fluor ® 488- or Alexa Fluor ® 555- conjugated secondary antibodies (Life Technologies). Cells were washed and nuclei were stained with Hoechst. Images were visualized with a Nikon inverted microscope.
  • the off-target sites were predicted using CCTop (Stemmer et ak, 2015).
  • the bait design, the enrichment of genomic DNA (library preparation), and NGS were conducted by Arbor Biosciences using myBaits ® custom target capture kit. Briefly, for each of the 648 predicted off-target sites, five RNA baits were designed across each off-target site and placed every ⁇ 26 bp, covering a 181-182 bp window.
  • RNA-gDNA hybrids were bound to streptavidin-coated beads and non-specific bonds were washed off.
  • the remaining gDNA libraries were amplified and sequenced by paired-end NGS using NovaSeq (Illumina).
  • % reads with altered sequence was found > 0 in WT as well as in all three engineered lines, the sequences were further inspected. In case the sequences of all three engineered lines matched the WT sequence, they were classified as SNP/PM; however, in case the sequences from the engineered cell lines deviated from the WT sequence, they were identified as editing events. Polymorphisms (PM) represent small deletions/insertions instead of single nucleotide polymorphisms (SNPs) observed already in the WT hPSCs, deviating from hg38.
  • SNPs single nucleotide polymorphisms
  • Betancur P.A., Abraham, B.J., Yiu, Y.Y., Willingham, S.B., Khameneh, F., Zamegar, M., Kuo, A.H., McKenna, K., Kojima, Y., Leeper, N.J., et al. (2017).
  • a CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer. Nat Commun 5, 14802.
  • HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795-799.
  • TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide. Curr Biol 8, 1-10.
  • Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.
  • H.G. (1996).
  • Nonclassical HLA-G molecules are classical peptide presenters. Curr Biol 6, 305-314.
  • HLA-G At the Interface of Maternal-Fetal Tolerance. Trends Immunol 38, 272-286. Glas, R., Franksson, L., Ohlen, C., Hoglund, P., Roller, B., Ljunggren, H.G., and Karre, K. (1992). Major histocompatibility complex class I-specific and - restricted killing of beta 2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 89, 11381-11385.
  • HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol 35, 765-772.
  • CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.
  • HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160, 4951-4960.
  • HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci U S A 95, 5199-5204.
  • CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.
  • mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med 12, 1316-1322.
  • the CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A 109, 6662-6667.

Abstract

L'invention concerne des cellules souches donnatrices universelles et leurs méthodes associées d'utilisation et de production. Les cellules souches donnatrices universelles selon l'invention sont utiles pour surmonter le rejet immunitaire dans des thérapies de transplantation à base de cellules. Selon certains modes de réalisation, les cellules souches donnatrices universelles révélées ici ont une expression modulée d'un ou de plusieurs antigènes leucocytaires humains MHC-I et MHC-II et d'un ou de plusieurs facteurs tolérogéniques.
PCT/US2020/018467 2019-02-15 2020-02-16 Cellules souches donnatrices universelles et méthodes associées WO2020168317A2 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
EA202192264A EA202192264A1 (ru) 2019-10-08 2020-02-16 Универсальные донорские стволовые клетки и соответствующие способы
AU2020223192A AU2020223192A1 (en) 2019-02-15 2020-02-16 Universal donor stem cells and related methods
SG11202108891QA SG11202108891QA (en) 2019-02-15 2020-02-16 Universal donor stem cells and related methods
JP2021547755A JP2022526218A (ja) 2019-02-15 2020-02-16 汎用ドナー幹細胞及び関連する方法
KR1020217029445A KR20210128440A (ko) 2019-02-15 2020-02-16 범용 공여자 줄기 세포 및 관련 방법
CA3130398A CA3130398A1 (fr) 2019-02-15 2020-02-16 Cellules souches donnatrices universelles et methodes associees
MX2021009842A MX2021009842A (es) 2019-02-15 2020-02-16 Células madre de donantes universales y métodos relacionados.
CN202080021860.7A CN113906048A (zh) 2019-02-15 2020-02-16 通用供体干细胞和相关方法
EP20755906.3A EP3924375A4 (fr) 2019-02-15 2020-02-16 Cellules souches donnatrices universelles et méthodes associées
BR112021016178A BR112021016178A2 (pt) 2019-02-15 2020-02-16 Células-tronco doadoras universais e métodos relacionados
IL285619A IL285619A (en) 2019-02-15 2021-08-15 Universal donor stem cells and related methods

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201916277913A 2019-02-15 2019-02-15
US16/277,913 2019-02-15
US201916596697A 2019-10-08 2019-10-08
US16/596,697 2019-10-08

Publications (3)

Publication Number Publication Date
WO2020168317A2 true WO2020168317A2 (fr) 2020-08-20
WO2020168317A3 WO2020168317A3 (fr) 2020-10-29
WO2020168317A8 WO2020168317A8 (fr) 2021-02-25

Family

ID=72045137

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/018467 WO2020168317A2 (fr) 2019-02-15 2020-02-16 Cellules souches donnatrices universelles et méthodes associées

Country Status (11)

Country Link
EP (1) EP3924375A4 (fr)
JP (1) JP2022526218A (fr)
KR (1) KR20210128440A (fr)
CN (1) CN113906048A (fr)
AU (1) AU2020223192A1 (fr)
BR (1) BR112021016178A2 (fr)
CA (1) CA3130398A1 (fr)
IL (1) IL285619A (fr)
MX (1) MX2021009842A (fr)
SG (1) SG11202108891QA (fr)
WO (1) WO2020168317A2 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021231712A1 (fr) * 2020-05-15 2021-11-18 Rxcell Inc. Cellules hypo-immunogènes et leurs utilisations dans des réponses immunitaires
CN114457021A (zh) * 2020-10-30 2022-05-10 未来智人再生医学研究院(广州)有限公司 一种表达cd47抗体的多能干细胞及其衍生物与应用
CN114525256A (zh) * 2020-10-30 2022-05-24 未来智人再生医学研究院(广州)有限公司 一种表达Siglec-15阻断物的多能干细胞或其衍生物及应用
WO2022125982A1 (fr) * 2020-12-11 2022-06-16 Intellia Therapeutics, Inc. Compositions et procédés pour réduire la mhc de classe ii dans une cellule
WO2022140587A1 (fr) * 2020-12-23 2022-06-30 Intellia Therapeutics, Inc. Compositions et procédés pour modifier génétiquement le ciita dans une cellule
WO2022146891A2 (fr) 2020-12-31 2022-07-07 Sana Biotechnology, Inc. Méthodes et compositions pour moduler une activité de car-t
US11459372B2 (en) 2020-11-30 2022-10-04 Crispr Therapeutics Ag Gene-edited natural killer cells
WO2022251367A1 (fr) 2021-05-27 2022-12-01 Sana Biotechnology, Inc. Cellules hypoimmunogènes comprenant hla-e ou hla-g génétiquement modifiés
WO2023019203A1 (fr) 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Systèmes inductibles pour modifier l'expression génique dans des cellules hypoimmunogènes
WO2023122337A1 (fr) 2021-12-23 2023-06-29 Sana Biotechnology, Inc. Lymphocytes t à récepteur antigénique chimérique (car) pour le traitement d'une maladie auto-immune et méthodes associées
WO2023154578A1 (fr) 2022-02-14 2023-08-17 Sana Biotechnology, Inc. Méthodes de traitement de patients présentant une thérapie préalable ayant échoué avec des cellules hypoimmunogènes
WO2023191063A1 (fr) * 2022-04-01 2023-10-05 株式会社Logomix Cellule appropriée pour l'ingénierie génique, l'ingénierie cellulaire et la médecine cellulaire, et son procédé de production
WO2024003349A1 (fr) 2022-07-01 2024-01-04 Novo Nordisk A/S Amélioration de la différenciation neuronale de cellules progénitrices neurales du mésencéphale ventral

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016183041A2 (fr) * 2015-05-08 2016-11-17 President And Fellows Of Harvard College Cellules souches de donneur universel et procédés associés
AU2016349504B2 (en) * 2015-11-04 2023-02-09 Fate Therapeutics, Inc. Genomic engineering of pluripotent cells
EA201991692A1 (ru) * 2017-01-13 2019-12-30 Дзе Риджентс Оф Дзе Юниверсити Оф Калифорния Иммуносконструированные плюрипотентные клетки

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021231712A1 (fr) * 2020-05-15 2021-11-18 Rxcell Inc. Cellules hypo-immunogènes et leurs utilisations dans des réponses immunitaires
CN114457021A (zh) * 2020-10-30 2022-05-10 未来智人再生医学研究院(广州)有限公司 一种表达cd47抗体的多能干细胞及其衍生物与应用
CN114525256A (zh) * 2020-10-30 2022-05-24 未来智人再生医学研究院(广州)有限公司 一种表达Siglec-15阻断物的多能干细胞或其衍生物及应用
US11459372B2 (en) 2020-11-30 2022-10-04 Crispr Therapeutics Ag Gene-edited natural killer cells
US11591381B2 (en) 2020-11-30 2023-02-28 Crispr Therapeutics Ag Gene-edited natural killer cells
WO2022125982A1 (fr) * 2020-12-11 2022-06-16 Intellia Therapeutics, Inc. Compositions et procédés pour réduire la mhc de classe ii dans une cellule
WO2022140587A1 (fr) * 2020-12-23 2022-06-30 Intellia Therapeutics, Inc. Compositions et procédés pour modifier génétiquement le ciita dans une cellule
US11802157B2 (en) 2020-12-31 2023-10-31 Sana Biotechnology, Inc. Methods and compositions for modulating CAR-T activity
WO2022146891A2 (fr) 2020-12-31 2022-07-07 Sana Biotechnology, Inc. Méthodes et compositions pour moduler une activité de car-t
US11965022B2 (en) 2020-12-31 2024-04-23 Sana Biotechnology, Inc. Methods and compositions for modulating CAR-T activity
WO2022251367A1 (fr) 2021-05-27 2022-12-01 Sana Biotechnology, Inc. Cellules hypoimmunogènes comprenant hla-e ou hla-g génétiquement modifiés
WO2023019203A1 (fr) 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Systèmes inductibles pour modifier l'expression génique dans des cellules hypoimmunogènes
WO2023122337A1 (fr) 2021-12-23 2023-06-29 Sana Biotechnology, Inc. Lymphocytes t à récepteur antigénique chimérique (car) pour le traitement d'une maladie auto-immune et méthodes associées
WO2023154578A1 (fr) 2022-02-14 2023-08-17 Sana Biotechnology, Inc. Méthodes de traitement de patients présentant une thérapie préalable ayant échoué avec des cellules hypoimmunogènes
WO2023191063A1 (fr) * 2022-04-01 2023-10-05 株式会社Logomix Cellule appropriée pour l'ingénierie génique, l'ingénierie cellulaire et la médecine cellulaire, et son procédé de production
WO2024003349A1 (fr) 2022-07-01 2024-01-04 Novo Nordisk A/S Amélioration de la différenciation neuronale de cellules progénitrices neurales du mésencéphale ventral

Also Published As

Publication number Publication date
KR20210128440A (ko) 2021-10-26
BR112021016178A2 (pt) 2021-11-03
WO2020168317A8 (fr) 2021-02-25
WO2020168317A3 (fr) 2020-10-29
EP3924375A4 (fr) 2023-03-22
CN113906048A (zh) 2022-01-07
CA3130398A1 (fr) 2020-08-20
IL285619A (en) 2021-09-30
JP2022526218A (ja) 2022-05-24
SG11202108891QA (en) 2021-09-29
AU2020223192A1 (en) 2021-09-09
MX2021009842A (es) 2021-12-10
EP3924375A2 (fr) 2021-12-22

Similar Documents

Publication Publication Date Title
WO2020168317A2 (fr) Cellules souches donnatrices universelles et méthodes associées
Dickinson et al. Graft-versus-leukemia effect following hematopoietic stem cell transplantation for leukemia
de Almeida et al. Immunogenicity of pluripotent stem cells and their derivatives
JP7123794B2 (ja) 免疫細胞組成物及びそれを使用する方法
Riolobos et al. HLA engineering of human pluripotent stem cells
JP6726656B2 (ja) クローディン６特異的免疫受容体およびｔ細胞エピトープ
EP3234144B1 (fr) Procédé pour l'élimination contrôlée de cellules thérapeutiques
JP2020040969A (ja) カスパーゼポリペプチドを使用して部分的なアポトーシスを誘導するための方法
JP2020191870A (ja) 治療用細胞のコントロールされた活性化または排除のための方法
CA3007473A1 (fr) Doubles commandes pour l'activation ou l'elimination de cellules therapeutiques
Shi et al. Generation of hypoimmunogenic human pluripotent stem cells via expression of membrane-bound and secreted β2m-HLA-G fusion proteins
CA3165076A1 (fr) Interrupteurs de securite pour la regulation de l'expression genique
JP2022511345A (ja) ＣＲＩＳＰＲ／Ｃａｓシステムに基づいた細胞の遺伝子編集の方法
Zhao et al. Strategies for genetically engineering hypoimmunogenic universal pluripotent stem cells
Meissner et al. Immune editing: overcoming immune barriers in stem cell transplantation
US20210171903A1 (en) Universal donor stem cells and related methods
US20230133564A1 (en) Cd3-fusion protein and uses thereof
EP4359541A2 (fr) Cellules ingéniérisées pour une thérapie
Bogomiakova et al. At home among strangers: is it possible to create hypoimmunogenic pluripotent stem cell lines?
Palma et al. HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy
Riddle et al. Rescue of cytotoxic function in the CD8α knockout mouse by removal of MHC class II
Anastasopoulou Targeting mutant p53 in spontaneous cancer by T cell receptor gene therapy
Merola Characterization of Human Endothelial Cells with CRISPR/Cas9-Mediated Complete Ablation of MHC Molecule Expression
MacNabb Dendritic Cells Prime Anti-tumor CD8 T Cell Responses through MHC-Dressing
Nagy Acceptance of allogeneic cell transplants without systemic immune suppression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20755906

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2021547755

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3130398

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021016178

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2020223192

Country of ref document: AU

Date of ref document: 20200216

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20217029445

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020755906

Country of ref document: EP

Effective date: 20210915

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20755906

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 112021016178

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20210816