Note: Descriptions are shown in the official language in which they were submitted.
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UNIVERSAL DONOR STEM CELLS AND RELATED METHODS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. Application No. 16/596,697, filed
on
October 8, 2019, which is a continuation of U.S. Application No. 16/277,913,
filed on
February 15, 2019, which claims the benefit of U.S. Provisional Application
No.
62/631,393, filed on February 15, 2018. The entire teachings of the above
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Therapies utilizing human pluripotent stem cell-derived cells for
transplantation have the potential to revolutionize the way diseases are
treated. A
major obstacle for their clinical translation is the rejection of allogeneic
cells by the
recipient's immune system. Strategies aiming at overcoming this immune barrier
include banking cells with defined HLA haplotypes (Nakajima et al., 2007;
Taylor et
al., 2005) and the generation of patient-specific induced pluripotent stem
cells (iPSCs)
(Takahashi et al., 2007; Yu et al., 2007). However, multiple limitations (de
Rham and
Villard, 2014; Tapia and Scholer, 2016) prohibit the broader use of these
approaches
and emphasize the need for "off-the-shelf' cell products that can be readily
administered to any patient in need.
SUMMARY OF THE INVENTION
Disclosed herein are efficient strategies to overcome immune rejection in cell-
based transplantation therapies by the creation of universal donor stem cell
lines.
Disclosed herein are stem cells comprising modulated expression of one or
more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic
factors relative to a wild-type stem cell.
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In some embodiments, the one or more MHC-I human leukocyte antigens are
selected from the group consisting of HLA-A, HLA-B, and HLA-C. In some
aspects,
the modulated expression of the one or more MHC-I human leukocyte antigens
comprises reduced expression of the one or more MHC-I human leukocyte
antigens.
In some embodiments, 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.
In some embodiments, the one or more MHC-II human leukocyte antigens are
selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. In some
aspects, the modulated expression of the one or more MHC-II human leukocyte
antigens comprises reduced expression of the one or more MHC-II human
leukocyte
antigens. In some embodiments, one or more indels were introduced into CIITA,
thereby modulating the expression of the one or more MHC-II human leukocyte
antigens.
In some embodiments, the cell does not express HLA-A, HLA-B, and HLA-C.
In certain aspects, the cell is an HLA-A-/-, HLA-C-/-, and CIITA'n''' cell.
In some embodiments, the one or more tolerogenic factors are selected from
the group consisting of HLA-G, PD-L1, and CD47. In certain aspects, the
modulated
expression of the one or more tolerogenic factors comprises increased
expression of
the one or more tolerogenic factors. In some embodiments, 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.
In some embodiments, the stem cell is an embryonic stem cell. In some
aspects, the stem cell is a pluripotent stem cell. In some embodiments, the
stem cell is
hypoimmunogenic. In some aspects, the stem cell is a human stem cell.
In some embodiments, 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.
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Also disclosed herein are stem cells that do not express HLA-A, HLA-B,
HLA-C, HLA-DP, HLA-DQ, and HLA-DR.
In some embodiments, the stem cell is a HLA-A-/-, HLA-C-/-, and
CIITAindel/indel cell. In some aspects, the stem cell expresses tolerogenic
factors HLA-
G, PD-L1, and CD47. In some embodiments, the tolerogenic factors are inserted
into
an AAVS1 safe harbor locus. In certain aspects, the tolerogenic factors
inhibit
immune rejection.
In some embodiments, the stem cell is an embryonic stem cell. In some
aspects, the stem cell is a pluripotent stem cell. In some embodiments, the
stem 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.
In some embodiments, the one or more MHC-I human leukocyte antigens are
selected from the group consisting of HLA-A, HLA-B, and HLA-C. In some
aspects,
the modulated expression of the one or more MHC-I human leukocyte antigens
comprises reduced expression of the one or more MHC-I human leukocyte
antigens.
In some embodiments, 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.
In some embodiments, the one or more MHC-II human leukocyte antigens are
selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR. In some
aspects, the modulated expression of the one or more MHC-II human leukocyte
antigens comprises reduced expression of the one or more MHC-II human
leukocyte
antigens. In some embodiments, one or more indels were introduced into CIITA,
thereby modulating the expression of the one or more MHC-II human leukocyte
antigens.
In some aspects, 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-/-, HLA-C-/-, and CIITAindevindel cell.
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In some embodiments, the one or more tolerogenic factors are selected from
the group consisting of HLA-G, PD-L1, and CD47. In some aspects, the modulated
expression of the one or more tolerogenic factors comprises increased
expression of
the one or more tolerogenic factors. In some embodiments, 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.
In some embodiments, the hypoimmunogenic stem cell retains pluripotency.
In some aspects, 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.
In some embodiments, 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. In some
aspects,
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. In some aspects, 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.
In some aspects, 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
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the stem cell is contacted with a Cas protein or a nucleic acid sequence
encoding the
Cas protein and a first pair of ribonucleic acids having sequences SEQ ID NOS:
1-2,
thereby editing the HLA-A gene to reduce or eliminate HLA-A surface expression
and/or activity in the stem cell, wherein the stem cell is contacted with a
Cas protein
or a nucleic acid sequence encoding the Cas protein and a second pair of
ribonucleic
acids having sequences SEQ ID NOS: 3-4, thereby editing the HLA-B gene to
reduce
or eliminate HLA-B surface expression and/or activity in the stem cell,
wherein the
stem cell is contacted with a Cas protein or a nucleic acid sequence encoding
the Cas
protein and a third 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, and wherein the stem cell is contacted with
a Cas
protein or a nucleic acid sequence encoding the Cas protein and a ribonucleic
acid
having 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 transplanting at least one
hypoimmunogenic stem cell into a patient, wherein the hypoimmunogenic stem
cell
comprises modulated expression of one or more MHC-I and MHC-II human
leukocyte antigens and one or more tolerogenic factors relative to a wild-type
stem
cell.
Also disclosed herein are stem cells. The stem cells may comprise reduced
expression of MHC-I and MHC-II human leukocyte antigens relative to 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.
In some embodiments, 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.
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In some embodiments, the tolerogenic factor is inserted into a safe harbor
locus of at
least one allele of the cell.
In some embodiments, the stem 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-Li.
Disclosed herein are stem cells that do not express HLA-A, HLA-B, HLA-C,
HLA-DP, HLA-DQ, and HLA-DR and expresses CD47. In some embodiments, the
cell is a CIITAindel/indel, HLA-A-/-, HLA-B-/-, and HLA-C-/- stem cell.
Also disclosed herein are methods of preparing hypoimmunogenic stem cells.
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.
In some embodiments, 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.
In some embodiments, the tolerogenic factor further comprises PD-Li and/or
HLA-G. In some embodiments, the hypoimmunogenic stem cell does not express
HLA-A, HLA-B, and HLA-C. In some embodiments, the hypoimmunogenic stem
cell does not express HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the
hypoimmunogenic stem cell does not express CIITA.
BRIEF DESCRIPTION OF THE DRAWINGS
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The patent or application file contains at least one drawing executed in
color.
Copies of this patent or patent application publication with color drawings
will be
provided by the Office upon request and payment of the necessary fee.
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. 1A 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. 1B 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 IFNy for 48 hrs before staining with the indicated antibodies.
FIG. 1D
shows targeting strategy of CIITA locus. Blue arrows indicate primers used for
PCR
.. and Sanger sequencing. FIG. 1E shows HLA-DR mean fluorescence intensity
(MFI)
in differentiated CD144+ WT and KO ECs. FIG. 1F provides a schematic
describing
the genotypes of WT, KO, KI-PHC, and KIPC cell lines. 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-Li 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-Li 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. 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
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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.
FIGS. 3A-3D demonstrate reduced T cell activities against KO and KI-PHC
cell lines in vitro. FIG. 3A provides scatterplots displaying the percent of
proliferating T cells in CD3+ (left panel, n=8 donors), CD4+ (middle panel,
n=6
donors), and CD8+ T cell populations (right panel, n=6 donors) when co-
cultured for
5 days with WT, KO, or KI ECs. 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. FIG. 3B provides a scatterplot displaying the percentage of
CD69+
(upper panel) and CD25+ cells (lower panel) in CD3+ (left panel), CD4+ (middle
panel), and CD8+ T cell populations (right panel) after a five-day co-culture
with WT,
KO, or KI ECs (n=11 donors in all plots). 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.
FIG. 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 graph
representing percent T cell cytotoxicity against WT, KO, and KI ECs (n=6
donors).
LDH release assay was performed and the percentage of T cell cytotoxicity from
each
donor was calculated. Paired one-way ANOVA followed by Tukey's multiple
comparison test. Data are mean s.e.m.; *p<0.05; **p<0.01.
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. Genotype of teratoma: WT (n=9), KO (n=7), KI-PHC
(n=6), and KI-PC (n=7). Ordinary one-way ANOVA followed by Tukey's multiple
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comparison test. Data are mean s.e.m.; *p<0.05. FIG. 4C shows percentage of
increased teratoma volume on day 0 of T cell injection compared to 2 days pre-
injection. Genotype of teratoma: WT (n=9), KO (n=7), KI-PHC (n=6), and KI-PC
(n=7). FIG. 4D shows relative hCD8 (left panel) and IL-2 (right panel) mRNA
expression in WT (n=8), KO (n=7), KI-PHC (n=6), and KI-PC (n=7) teratomas
harvested on day 8 post-T cell injection. The expression was normalized to
RPLPO.
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. FIG. 5A provides a scatterplot of NK cell degranulation
against WT, KO, or KI-PHC VSMCs (n=7 donors). The percentage of degranulating
NK cells was plotted as % CD107a-expressing CD56+ cells for each donor. NK
cells
cultured alone were used as negative control; NK cells treated with
PMA/ionomycin
served as positive control. Paired one-way ANOVA followed by Tukey's multiple
comparison test. Data are mean s.e.m.; **p<0.01. FIG. 5B provides a bar
graph
representing the percentage of NK cytotoxicity against WT, KO, and KI-PHC
VSMCs from one representative donor at the indicated effector/target (E/T)
ratios
(n=3 replicates). LDH release assay was performed and the % NK cytotoxicity
was
calculated as specific lysis of NK cell-killed VSMCs relative to maximum cell
lysis.
Unpaired one-way ANOVA followed by Tukey's multiple comparison test. Data are
mean s.d.; *p<0.05; ***p<0.001. FIG. 5C provides time-lapse plots of
macrophage
phagocytosis assay (n=5 monocyte donors). 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 mm using Celldiscover 7 live cell imaging
system. Total integrated fluorescence intensity of pHrodored+ phagosomes per
image
was analyzed. Data are mean s.e.m. FIG. 5D provides scatterplots of
macrophage
phagocytosis assay at 4 hr co-incubation (n=9 monocyte donors, three
independent
experiments). The experimental conditions were the same as in FIG. 5C. Paired
one-
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way ANOVA followed by Tukey's multiple comparison test. Data are mean
s.e.m.;
*p<0.05; **p<0.01. VSMC = vascular smooth muscle cells; NK = natural killer
cells.
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. 1B. 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. 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 AAVS1 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 Ia 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-Li 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-Li 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
differentiated WT, KO, and KI-PHC VSMCs upon IFN-y stimulation. Gray,
isotype; colored, antibodies. FIG. 7H shows relative HLA-E mRNA expression in
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differentiated WT, KO, and KI-PHC VSMCs with or without IFN-y
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+ (middle
panel), and CD8+ (bottom panel). T cells cultured alone were used as negative
control;
T cells treated with CD3/CD28 beads served as positive control. FIG. 10C shows
doxycycline-inducible PD-Li expression in WT VSMCs. FIG. 10D provides a
.. scatterplot of percent proliferating T cells in CD3+ (left panel), CD4+
(middle panel),
and CD8+ T cell populations (right panel) co-cultured for 7 days with VSMCs in
the
presence or absence of doxycycline-induced PD-Li expression (n=4 donors). 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.
VSMCs were either pretreated with staurosporine or left untreated. The images
represent overlays of bright field and red channel and the fluorescent
phagosomes are
highlighted after masking by the ZEN imaging analysis software. Scale bars,
200 pm.
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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. 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 HUES8.
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-Li 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.
FIGS. 14A-14B demonstrate functional immune-silent cells for
transplantation. 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). For the 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
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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. For the NK
cell degranulation assay (bottom panel), an HLA-deficient VSMC (D1, F2)
trigger
enhanced NK cell degranulation when compared to WT cells. 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
transplanted with ES cells of the indicated genotype, and allowed to form
teratoma. 4-
6 weeks post transplantation teratoma size and consistency were scored in a
blinded
manner. While the WT teratoma show hallmarks of rejection, growth of the NLCR5-
/-
.. CIITA-/- and B2M-/-CIITA-/- stem cells was found less restricted,
suggesting they
are immune-protected. FIG. 15B shows introduction of an inducible Caspase9
(iCasp9) killing switch can ablate cells upon treatment with the CID
dimerizer.
Cartoon of the iCasp9 killing switch (left). Dose titration of the CID
dimerizer and
time course in transiently transfected 293T cells (right). Ultimately, the
iCasp9 killing
switch will be integrated into a safe harbor locus of the modified, immune
silent stem
cells.
DETAILED DESCRIPTION OF THE INVENTION
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.
As used herein to characterize a cell, the term "hypoimmunogenic" generally
means that such cell is less prone to immune rejection by a subject into which
such
cells are transplanted. For example, relative to an unaltered wild-type cell,
such a
hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
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70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection by a
subject into which such cells are transplanted. In some aspects, genome
editing
technologies (e.g., the CRISPR/Cas or TALEN systems) are used to modulate
(e.g.,
reduce or eliminate) the expression of MHC-I and MHC-II genes.
In certain embodiments, 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. Such CRISPR/Cas systems can
employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;1(6)e60).
In
some embodiments, the CRISPR/Cas system is a CRISPR type I system. In some
embodiments, the CRISPR/Cas system is a CRISPR type II system. In some
embodiments, the CRISPR/Cas system is a CRISPR 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. In
some
embodiments, the target polynucleotide sequence in a cell (e.g., ES cells or
iPSCs) is
altered to produce a mutant cell. As used herein, a "mutant cell" generally
refers to a
cell with a resulting genotype that differs from its original genotype or the
wild-type
cell. In some instances, a "mutant cell" exhibits a mutant phenotype, for
example
when a normally functioning stem gene is altered using the CRISPR/Cas systems.
In
some embodiments, 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).
In some
embodiments, 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).
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In some embodiments, the alteration is an indel. As used herein, "indel"
refers
to a mutation resulting from an insertion, deletion, or a combination thereof.
As will
be appreciated by those skilled in the art, 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. In some embodiments, the alteration is a point mutation. As
used
herein, "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.
In some embodiments, the alteration results in a knock out of the target
.. polynucleotide sequence or a portion thereof. For example, 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).
As used herein, "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.
In some embodiments, 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"
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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.
The term "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.
In some embodiments, the alteration is a homozygous alteration. In some
embodiments, the alteration is a heterozygous alteration.
In some embodiments, the alteration results in 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. For example, 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.
In some
instances, 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. In certain embodiments, the efficiency of alteration is at least
about 5%.
In certain embodiments, 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
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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%.
In some embodiments, the target polynucleotide sequence is a genomic
sequence. In some embodiments, the target polynucleotide sequence is a human
genomic sequence. In some embodiments, the target polynucleotide sequence is a
mammalian genomic sequence. In some embodiments, the target polynucleotide
sequence is a vertebrate genomic sequence.
In some embodiments, 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. In some embodiments, 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. As used herein, "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.,
phosphorylated, glycated, glycosolated, etc.) and amino acid analogs.
Exemplary
polypeptides or proteins include gene products, naturally occurring proteins,
homologs, paralogs, fragments and other equivalents, variants, and analogs of
the
above.
In some embodiments, a Cas protein comprises one or more amino acid
substitutions or modifications. In some embodiments, the one or more amino
acid
substitutions comprise a conservative amino acid substitution. In some
instances,
substitutions and/or modifications can prevent or reduce proteolytic
degradation
and/or extend the half-life of the polypeptide in a cell. In some embodiments,
the Cas
protein can comprise a peptide bond replacement (e.g., urea, thiourea,
carbamate,
sulfonyl urea, etc.). In some embodiments, the Cas protein can comprise a
naturally
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occurring amino acid. In some embodiments, the Cas protein can comprise an
alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine,
phosphoserine, etc.). In some embodiments, a Cas protein can comprise a
modification to include a moiety (e.g., PEGylation, glycosylation, lipidation,
acetylation, end-capping, etc.).
In some embodiments, 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. In some embodiments, 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. In
some
embodiments, 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. In some embodiments, 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. In
some embodiments, a Cas protein comprises a Cas protein of the Dvulg subtype
(also
known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csdl,
Csd2, and Cas5d. In some embodiments, 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. In some
embodiments, a
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. In
some
embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, the Cas protein is Cas9 protein or a functional portion
thereof. In some embodiments, the Cas protein is Cas9 from any bacterial
species or
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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) 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.
In some embodiments, 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.
As used herein, "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. In some embodiments, 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.
In
some embodiments, 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.
It should be appreciated that the present invention contemplates various ways
of contacting a target polynucleotide sequence with a Cas protein (e.g.,
Cas9). In
some embodiments, exogenous Cas protein can be introduced into the cell in
polypeptide form. In certain embodiments, Cas proteins can be conjugated to or
fused
to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein,
"cell-
penetrating polypeptide" and "cell-penetrating peptide" refers to a
polypeptide or
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peptide, respectively, which facilitates the uptake of molecule into a cell.
The cell-
penetrating polypeptides can contain a detectable label.
In certain embodiments, 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. In some embodiments, 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 Bio1.2010;5(8):747-
52). In
certain embodiments, the Cas protein can be fused to a protein transduction
domain
(PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat,
oligoarginine,
and penetratin. In some embodiments, the Cas protein comprises a Cas
polypeptide
fused to a cell-penetrating peptide. In some embodiments, the Cas protein
comprises
a Cas polypeptide fused to a PTD.
In some embodiments, the Cas protein 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. In some embodiments, the nucleic acid
comprises
DNA. In some embodiments, the nucleic acid comprises a modified DNA, as
described herein. In some embodiments, the nucleic acid comprises mRNA. In
some
embodiments, the nucleic acid comprises a modified mRNA, as described herein
(e.g.,
a synthetic, modified mRNA).
In some embodiments, 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., lentiviral transduction).
In some embodiments, the Cas protein is complexed with one to two
ribonucleic acids. In some embodiments, the Cas protein is complexed with two
ribonucleic acids. In some embodiments, the Cas protein is complexed with one
ribonucleic acid. In some embodiments, the Cas protein is encoded by a
modified
nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
The methods of the present invention contemplate the use of any ribonucleic
acid that is capable of directing a Cas protein to and hybridizing to a target
motif of a
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target polynucleotide sequence. In some embodiments, at least one of the
ribonucleic
acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic
acids comprises CRISPR RNA (crRNA). In some embodiments, 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. In some embodiments, 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.
In some
embodiments, 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
CRISPR/Cas system employed, and the sequence of the target polynucleotide, as
will
be appreciated by those skilled in the art. The one to two ribonucleic acids
can also
be selected to minimize hybridization with nucleic acid sequences other than
the
target polynucleotide sequence. In some embodiments, 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. In
some embodiments, 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.
In some embodiments, 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.
In some embodiments, at least one of the one to two ribonucleic acids
comprises a sequence with a single nucleotide mismatch to a sequence selected
from
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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.
In some embodiments, 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. In some embodiments, one or two
ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to
sequences on the same strand of a target polynucleotide sequence. In some
embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary
to
and/or hybridize to sequences on the opposite strands of a target
polynucleotide
sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide
RNAs)
are not complementary to and/or do not hybridize to sequences on the opposite
strands of a target polynucleotide sequence. In some embodiments, the one or
two
ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to
overlapping target motifs of a target polynucleotide sequence. In some
embodiments,
the one or two ribonucleic acids (e.g., guide RNAs) are complementary to
and/or
hybridize to offset target motifs of a target polynucleotide sequence.
In some embodiments, the target motif is a 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.
In some embodiments, 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. Those skilled in the
art will
appreciate that a variety of techniques can be used to select suitable target
motifs for
minimizing off-target effects (e.g., bioinformatics analyses). In some
embodiments,
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
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recognized by the Cas protein which flank a mutant allele located between the
target
motifs.
In some aspects, 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.). In
some embodiments, 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). In some embodiments, 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). In still other embodiments, 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.
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). As used
herein, a
"subject" means a human or animal. In certain embodiments, the subject is a
human.
In certain embodiments, the subject is an adolescent. In certain embodiments,
the
subject is treated in vivo, in vitro and/or in utero. In certain aspects, 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. In some aspects,
the
universal stem cells are transplanted into a subject.
Provided herein are novel cells, compositions and methods that are useful for
addressing such HLA-based immune rejection of transplanted cells.
Ablation of MHC Class I and MHC Class II Genes
In certain aspects, the inventions disclosed herein relate to genomic
modifications of one or more targeted polynucleotide sequences of the stem
cell
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genome that regulates the expression of MHC-I and/or MHC-II human leukocyte
antigens. In some aspects, a genetic editing system is used to modify one or
more
targeted polynucleotide sequences. In some aspects, 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.
In certain embodiments, HLA expression is interfered with. In some aspects,
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). In some aspects multiple HLAs may
be
targeted at the same time. For example, HLA-B and HLA-C are adjacent and may
be
targeted simultaneously. In some aspects 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. In some aspects a 13 kb deletion of a stem cells genome using
CRISPR/Cas knocks out HLA-A, as well as the promoter of the gene.
In certain aspects, 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. For example, in certain aspects, 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. In some aspects, one
or more of HLA-A, HLA-B and HLA-C may be "knocked-out" 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. In some aspects,
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. In some aspects, 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
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CIITA (e.g., targeting exon 1) may exhibit reduced or eliminated expression of
HLA-
DP, HLA-DQ, and/or HLA-DR.
In some aspects, the present disclosure provides a stem cell (e.g.,
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.
In certain aspects, 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.
In some aspects, the present disclosure provides a stem cell (e.g.,
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.
In certain aspects, 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
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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.
In some aspects, the present disclosure provides a stem cell (e.g.,
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.
In certain aspects, 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.
In certain aspects, 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. In some aspects 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.
In certain aspects, 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
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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. In some aspects 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.
Insertion of Tolerogenic Factors
In certain embodiments, one or more tolerogenic factors can be inserted or
reinserted into genome-edited stem cell lines to create immune-privileged
universal
donor stem cells. In certain embodiments, the universal stem cells disclosed
herein
have been further modified to express one or more tolerogenic factors.
Exemplary
tolerogenic factors include, without limitation, one or more of HLA-G, PD-L1,
and
CD47. The expression of such tolerogenic factors may inhibit immune rejection.
The present inventors have used genome editing systems, such as the
CRISPR/Cas-assisted homology directed repair (HDR) system, to facilitate the
insertion of tolerogenic factors into a safe harbor locus, such as the AAVS1
locus, to
actively inhibit immune rejection. In some aspects a donor plasmid comprises a
HLA-G expression cassette. In some aspects a donor plasmid comprises a PD-Li
expression cassette. In some aspects a donor plasmid comprises a CD47
expression
cassette. In certain aspects a donor plasmid comprises a PD-L1, HLA-G, and
CD47
expression cassette. In certain aspects a donor plasmid comprises a PD-Li 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). In certain
aspects 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:
8.
In some aspects, the present disclosure provides a stem cell (e.g.,
hypoimmunogenic stem cell) or population thereof comprising a genome in which
the
stem cell genome has been modified to express HLA-G. In some aspects, the
present
disclosure provides a method for altering a stem cell genome to express HLA-G.
In
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certain aspects 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.
In some aspects, the present disclosure provides a stem cell (e.g.,
hypoimmunogenic stem cell) or population thereof comprising a genome in which
the
.. stem cell genome has been modified to express PD-Li. In some aspects, the
present
disclosure provides a method for altering a stem cell genome to express PD-Li.
In
certain aspects at least one ribonucleic acid or at least one pair of
ribonucleic acids
may be utilized to facilitate the insertion of PD-Li into a stem cell line.
In some aspects, the present disclosure provides a stem cell (e.g.,
hypoimmunogenic stem cell) or population thereof comprising a genome in which
the
stem cell genome has been modified to express CD-47. In some aspects, the
present
disclosure provides a method for altering a stem cell genome to express CD-47.
In
certain aspects 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.
In some aspects, 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.
In some aspects, the present disclosure provides a method for altering a stem
cell
genome to express PD-L1, HLA-G, and CD47.
In some aspects, 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-Li and CD47. In
some
.. aspects, the present disclosure provides a method for altering a stem cell
genome to
express PD-Li and CD47.
Universal Stem Cells
In certain aspects, 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. In certain aspects the universal stem cells
are HLA-
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HLA-B-/-, HLA-C-/-, and CIITAindelhndel cells that exhibit increased
expression of
HLA-G, PD-L1, and CD47.
In some aspects the stem cells (e.g., the universal stem cells) described
herein
exhibit one or more features. For example, 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. For example, the universal stem
cells
may retain expression of one or more of NANOG, OCT4, SSEA3, and TRA-1-60. In
some aspects the universal stem cells are differentiated into the three germ
layers
(e.g., ectoderm, mesoderm, and endoderm) and maintain expression of all
lineage
markers.
In some aspects the universal stem cells demonstrate reduced T cell-mediated
adaptive immune responses. For example, T cells (e.g., CD4+ and CD8+ T cells)
exhibit reduced priming and activation against the universal stem cells. In
addition, 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. In some aspects, the expression of
PD-Li
further suppresses activation of CD8+ T cells.
In some embodiments 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. In some embodiments the universal
stem
cells exhibit reduced macrophage engulfment. Overexpression of CD47 and/or
expression of PD-Li in the universal cells may minimize or inhibit macrophage
engulfment of the universal cells.
Some Definitions
Unless otherwise defined herein, scientific and technical terms used in
connection with the present application shall have the meanings that are
commonly
.. understood by those of ordinary skill in the art. Further, unless otherwise
required by
context, singular terms shall include pluralities and plural terms shall
include the
singular.
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As used herein the term "comprising" or "comprises" is used in reference to
compositions, methods, kits and respective component(s) thereof, that are
essential to
the invention, yet open to the inclusion of unspecified elements, whether
essential or
not.
As used herein 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
characteristic(s) of that embodiment of the invention.
The term "consisting of' refers to compositions, methods, kits and respective
components thereof as described herein, which are exclusive of any element not
recited in that description of the embodiment.
Other than in the operating examples, or where otherwise indicated, all
numbers expressing quantities of ingredients or reaction conditions used
herein should
be understood as modified in all instances by the term "about." The term
"about"
when used in connection with percentages may mean 1%.
The singular terms "a," "an," and "the" include plural referents unless
context
clearly indicates otherwise. Similarly, the word "or" is intended to include
"and"
unless the context clearly indicates otherwise. It is further to be understood
that all
base sizes or amino acid sizes, and all molecular weight or molecular mass
values,
given for nucleic acids or polypeptides are approximate, and are provided for
description. Although methods and materials similar or equivalent to those
described
herein can be used in the practice or testing of this disclosure, suitable
methods and
materials are described below. The term "comprises" means "includes." The
abbreviation, "e.g." is derived from the Latin exempli gratia, and is used
herein to
indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous
with
the term "for example."
The entire teachings of PCT application PCT/U52016/031551, filed on May 9,
2016, are incorporated herein by reference. All other patents and publications
identified are expressly incorporated herein by reference for the purpose of
describing
and disclosing, for example, the methodologies described in such publications
that
might be used in connection with the disclosure. These publications are
provided
solely for their disclosure prior to the filing date of the present
application. Nothing
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in this regard should be construed as an admission that the inventors are not
entitled to
antedate such disclosure by virtue of prior invention or for any other reason.
All
statements as to the date or representation as to the contents of these
documents is
based on the information available to the applicants and does not constitute
any
admission as to the correctness of the dates or contents of these documents.
To the extent not already indicated, it will be understood by those of
ordinary
skill in the art that any one of the various embodiments herein described and
illustrated may be further modified to incorporate features shown in any of
the other
embodiments disclosed herein.
The following example illustrates some embodiments and aspects of the
invention. It will be apparent to those skilled in the relevant art that
various
modifications, additions, substitutions, and the like can be performed without
altering
the spirit or scope of the invention, and such modifications and variations
are
encompassed within the scope of the invention as defined in the claims which
follow.
The following examples do not in any way limit the invention.
EXEMPLIFICATION
Therapies utilizing human pluripotent stem cell-derived cells for
transplantation have the potential to revolutionize the way diseases are
treated. A
major obstacle for their clinical translation is the rejection of allogeneic
cells by the
recipient's immune system. Strategies aiming at overcoming this immune barrier
include banking cells with defined HLA haplotypes (Nakajima et al., 2007;
Taylor et
al., 2005) and the generation of patient-specific induced pluripotent stem
cells (iPSCs)
(Takahashi et al., 2007; Yu et al., 2007). However, multiple limitations (de
Rham and
Villard, 2014; Tapia and Scholer, 2016) prohibit the broader use of these
approaches
and emphasize the need for "off-the-shelf' cell products that can be readily
administered to any patient in need. As a first step to generate such a
universal stem
cell product, ablating HLA class I is necessary to prevent the presentation of
cellular
peptides to cytotoxic CDS+ T cells, given that HLA class I molecules are
expressed in
virtually all nucleated cells. Moreover, 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
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(APCs) and endothelial cells (ECs) upon IFNy stimulation (Ting and Trowsdale,
2002). Recently, the power of 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 al., 2014;
Mattapally
et al., 2018; Meissner et al., 2014; Riolobos et al., 2013; Wang et al.,
2015), and to
eliminate HLA class II expression by targeting its transcriptional master
regulator,
CIITA (Chen et al., 2015; Mattapally et al., 2018). However, the deletion of
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 al., 2017; Lee et al., 1998b). Moreover, it has been found that
B2M-
deficient cells are still rejected by allogeneic CD8+ T cells (Glas et al.,
1992).
Therefore, 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.
Other approaches that have been explored to create "off-the-shelf' cell
products include the expression of co-inhibitory molecules and the blocking of
co-
stimulatory signals required for full T cell activation beyond HLA-T cell
receptor
(TCR) engagement. For example, ectopic expression of the T cell checkpoint
inhibitors PD-Li and CTLA-41g has been shown to protect stem cells from
rejection
in a humanized mouse model (Rong et al., 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 al., 2007). Moreover, CTLA-41g can also impair T regulatory cell (Treg)
homeostasis and function, possibly jeopardizing the establishment of
operational
immune tolerance (Bour-Jordan et al., 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). Recently, Gornalusse et al. expressed a B2M-HLA-E fusion construct in
B2M-
deficient cells to overcome NK cell-mediated lysis (Gornalusse et al., 2017).
However, this approach does not address NK cells lacking NKG2A, an inhibitory
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receptor for HLA-E, whose reactivity could still be concerning (Braud et al.,
1998a;
Pegram et al., 2011). Therefore, 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. Moreover, 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). However, this approach has
not yet
been explored to protect hPSCs and their differentiated derivatives from
macrophage
engulfment. Furthermore, a convincing strategy to target both adaptive and
innate
immunity is yet to be proposed.
Here, it is demonstrated that the CRISPR/Cas9 system can be used to
selectively excise the genes encoding the polymorphic HLA class I members, HLA-
A/-B/-C, from the genome of hPSCs. Moreover, its multiplexing capacity allows
for
the simultaneous ablation of HLA class II gene expression using a single guide
RNA
targeting C//TA. The resulting polymorphic HLA-deficient, "immune-opaque"
cells
were further modified to express the immunomodulatory factors PD-L1, HLA-G and
CD47, which target immune surveillance by T cells, NK cells, and macrophages,
respectively, further 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.
Results
Genome Editing Ablates Polymorphic HL4-A1-B/-C and HLA class II Expression
Given that the human MHC class I genes HLA-A, HLA-B, and HLA-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. Thus, 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). In the HLA locus, HLA-B and HLA-C are
adjacent, whereas HLA-A is located nearer the telomere. To simultaneously
knock
out the adjacent HLA-B and HLA-C genes, two sgRNAs were designed at each site,
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upstream of HLA-B and downstream of HLA-C (FIG. 1A). The predicted 95 kb
deletion also includes the promoters of the two genes, defined as H3K27Ac-
positive
areas on the UCSC Genome Browser. To knock out the entire HLA-A gene, one
sgRNA was designed upstream and another sgRNA downstream of HLA-A (FIG.
1B). 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 -Al- 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. 1D) (Ding et al., 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). To demonstrate that targeting CIITA
resulted in
loss of HLA class II expression, both WT and KO hPSCs were differentiated into
endothelial cells (ECs) using a previously published protocol (Patsch et al.,
2015). Of
note, 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). Importantly, induction of
HLA-DR
expression upon IFNy stimulation was abolished in KO ECs (FIG. 1E). The KO
hPSC
clone with a genotype of HLA-A-/-HLA-B-/-HLAC-/-CIITAindel/indel was
generated following the workflow depicted in FIG. 6F. Taken together, 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.
Knock-in of Immunomodulatory Factors into HLA Knockout Cell Line
It was hypothesized that ablating the polymorphic HLA class Ia and class II
molecules would eliminate T cell-mediated adaptive immune rejection. However,
HLA knockout cells would likely still be susceptible to innate immune cells
involved
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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). Thus, failure to express any HLA
class I other
than HLA-E may render donor cells vulnerable to NK cell-mediated lysis. To
protect
the engineered cells from NK cells, it was sought to introduce HLA-G into HLA
knockout cells. 2) Macrophages are attracted by cytokines secreted at the site
of
engraftment and are primed to phagocytose foreign cells by antibody binding.
It has
been well documented that 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 al., 2017; Jaiswal et al., 2009; Willingham et al.,
2012; Zhao
et al., 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 al., 1996), which would potentially re-expose the cell lines to CD8+
T cell
immune surveillance. Furthermore, y61' cells can directly recognize antigens
and
initiate a cytotoxic response (Vantourout and Hayday, 2013). To counteract any
residual T cell response, it was decided to knock in PD-L1, a T cell
checkpoint
inhibitor that engages the PD-1 receptor on activated T cells, directly
suppressing T
cell activities (Riley, 2009). Moreover, PD-Li expression may also contribute
to
protecting transplanted cells from innate immune rejection by inhibiting PD-1+
NK
cells (Beldi-Ferchiou et al., 2016; Della Chiesa et al., 2016) and PD-1+
macrophages
.. (Gordon et al., 2017).
To avoid random integration and positional effects on transgene expression, it
was sought to knock-in the immunomodulatory factors into the AAVS1 safe harbor
locus (Sadelain et al., 2011). Two donor plasmids were designed, one
containing a
PD-Li; HLA-G; CD47 expression cassette and another one containing a PD-Li;
CD47 expression cassette, both driven by a CAGGS promoter flanked by arms
homologous to the AAVS1 locus (FIG. 1G). The donor plasmids were
electroporated
together with a sgRNA targeting the AAVS1 locus into the HLA-A-/-HLAB-/-HLA-C-
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/-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.
1H-1I), and a second one named KI-PC that expressed PD-Li and displayed
elevated
CD47 level (FIG. 1J and FIG. 7A). Surface HLA-A2 levels were checked by flow
cytometry in both KI clones and confirmed HLA class Ia ablation (FIG. 7B). KI-
PHC
and KI-PC hPSCs were differentiated into CD144+ ECs (FIG. 7C), and no HLA-DR
expression was observed by flow cytometry following IFNy stimulation (FIG.
7D).
Thus, immunomodulatory factors were successfully inserted into the AAVS1 safe
harbor locus of HLA class Ia and II null cells. Altogether, three engineered
hPSC
lines: KO, KI-PHC, and KI-PC (FIG. 1F) were generated.
Next, it was sought to confirm the transgene expression as well as HLA-E
expression in derivatives of the engineered hPSC lines. For this purpose, 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).
In KI-PHC VSMCs, a subpopulation with modestly higher expression of PD-Li and
HLA-G was observed, compared to WT VSMCs, and a major population displaying
significantly elevated levels of PD-Li and HLA-G (FIG. 7F). However, 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). Similarly, a small
subpopulation with
modestly higher, and a major population with highly elevated levels of PD-Li
and
CD47 in KI-PC VSMCs was observed, compared to WT VSMCs (FIG. 7F).
When WT VSMCs were stimulated with IFNy, they drastically upregulated
HLA-E surface expression. In contrast, 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). Surprisingly, surface HLA-E expression
of
KI-PHC VSMCs was not restored by HLA-G expression (FIG. 7G). Nevertheless,
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
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products to compensate for the reduction of HLA-E surface expression in an HLA-
A/-
B/-C null background.
KO and KI Cell Lines Retain Pluripotency and Differentiation Potential
To assess whether the engineered hPSC lines retained pluripotency, expression
of NANOG, OCT4, SSEA3, SSEA4, and TRA-1-60 was assessed by
immunofluorescence on KO, KI-PHC and KI-PC hPSCs and found equivalent to that
of unmodified hPSCs (FIG. 2A). In addition, KO, KI-PHC and KI-PC hPSCs were
differentiated into the three germ layers. qRT-PCR was carried out to examine
the
expression of ectoderm, mesoderm, and endoderm markers and compared to the
three
germ layers derived from unmodified hPSCs. All of the lineage markers analyzed
were found expressed in their respective germ layer cells (FIG. 2B). In
addition, the
KO, KI-PHC, and KI-PC hPSCs displayed a normal karyotype (FIG. 2C). Thus,
despite multipe rounds of genetic modification, these engineered hPSC lines
maintained pluripotency, performed tri-lineage differentiation, and retained a
normal
karyotype.
To analyze potential off-target effects of the sgRNAs used to engineer the
hPSC lines, 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 HLA-H (HFE), which displayed a perfect match
to the
sgRNA upstream of HLA-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. Following
enrichment by specifically designed RNA baits, for each predicted off-target
site, the
enriched DNA fragments were sequenced by next generation sequencing (NGS).
Sequence reads of each cell line were aligned and compared to the hg38 genomic
reference sequence, and the percentages of reads with altered sequences were
calculated. As a result, besides 12 naturally occurring SNP/polymorphic sites
identified, HLA-H (HFE) was confirmed as an off-target in all three cell
lines.
Moreover, an intronic off-target site was detected in TRAF3 in all three cell
lines
resulting from targeting HLA-C, as well as an intronic off-target site in
CPNE5 in the
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KI-PC cell line as a result of the AAVS1 sgRNA (FIG. 2E and FIG. 9).
Altogether,
although three off-target events were detected, 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.
Reduced T Cell Responses Against KO and KI Cell Lines
Given that removing polymorphic HLA class Ia expression is expected to
eliminate T cell-mediated adaptive immune responses, it was next sought to
investigate T cell activities in co-cultures with the engineered cell lines.
In addition to
the KO cells, the KI-PHC cells were also used to address whether the
expression of
the T cell checkpoint inhibitor PD-Li would further suppress T cell activity.
Four
separate in vitro T cell immunoassays were performed: T cell proliferation,
activation,
cytokine secretion, and killing assays. Since HLA I expression is modest in
hPSCs (de
Almeida et al., 2013; Drukker et al., 2002), the engineered as well as WT
hPSCs were
differentiated into ECs, which express both HLA I and II following IFNy
stimulation,
or into VSMCs, which only express HLA I, before being used in the respective
immunoassays.
For T cell proliferation assays, WT, KO, and KI-PHC ECs were pre-treated
with IFNy 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. 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. 3A, left panel). CD4+ T cells followed a similar pattern, with WT ECs
(5.03%
0.89% SEM) inducing more CD4+ T cell proliferation than KO ECs (3.58%
0.86% SEM) or KI-PHC ECs (3.49% 0.83% SEM) (FIG. 3A, middle panel).
Moreover, CD8+ 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).
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Importantly, when compared to co-cultures with KO ECs, CD8+ T cells
proliferated
significantly less in the presence of KI-PHC ECs (FIG. 3A, right panel),
indicating
that CD8+ T cell activation was suppressed even further by overexpression of
PD-Li
in an HLA null background. To further investigate the suppressive role of PD-
Li
during the responses of different T cell subpopulations, an inducible PD-L1-
expressing hPSC line was generated and differentiated into ECs before
conducting a T
cell proliferation assay. It was found that only CD8+, not CD4+, T cell
proliferation
was reduced in the presence of PD-Li-expressing ECs, when compared to WT ECs,
arguing for a specific inhibitory effect of PD-Li on the CD8+ T cell subset
(FIGS.
10C-10D).
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. 3B). It also found
that,
when co-cultured with WT ECs, a higher percentage of CD25+ cells was observed
in
the CD4+ cell population, whereas a higher percentage of CD69+ cells was
observed
in the CD8+ cell population. However, a significantly reduced expression of
activation markers in T cells against KI-PHC ECs when compared to KO ECs was
not
observed.
Next, the levels of the T cell effector cytokines IFNy and IL-10 secreted into
the medium over the course of a five-day T cell-EC co-culture were examined.
Compared to the levels of IFNy and IL-10 observed in media following exposure
to
WT ECs (4747 556.1 SD; 54.56 17.22 SD), the levels of both cytokines were
lower in media when T cells were exposed to either the KO (3214 180.5 SD;
5.09
0.16 SD) or KI-PHC ECs (2635 132.9 SD; 3.56 0.63 SD), indicating reduced
cytokine secretion from T cells against KO or KI-PHC cell lines (FIG. 3C).
To quantify T cell killing, lactate dehydrogenase (LDH) released from
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
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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-Li in KI-PHC VSMCs, consistent with the results of the CD8+ T cell
proliferation assay. Collectively, the observations in the T cell immunoassays
demonstrate reduced CD4+ and CD8+ T cell priming against KO and KI-PHC cell
lines as a result of the removal of HLA I and II molecules. CD8+ T cell
activation was
further suppressed by the expression of PD-Li in the KIPHC cell line.
To assess T cell responses in vivo, WT and the engineered hPSCs were
transplanted subcutaneously into immunodeficient mice and allowed to form
teratomas over the course of 4-6 weeks. Pre-sensitized allogeneic CD8+ T cells
were
then adoptively transferred via tail vein injection and teratoma growth was
monitored
for an additional 8 days (FIG. 4A). As measured by CD69 and PD-1 expression of
CD8+ T cells pre- and post-priming, the T cells used for injection were
activated
(CD69+) and without signs of exhaustion (PD-1+) following sensitization (FIG.
11A).
In agreement with the hypothesis that only the WT cells will be rejected, 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). In
addition,
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 CD8
and IL-
2 (FIG. 4D), as well as by histology (FIG. 4E). Together, these observations
suggest
that removal of the polymorphic HLA molecules from the cell surface of
transplanted
cells can effectively block T cell-mediated rejection in vivo, matching the in
vitro
observations.
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K1 Cell Lines are Protected from NK Cell and Macrophage Responses
Due to the lack of HLA Ia 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. To test the hypothesis,
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. 11B). Of note, NK cell degranulation in the presence of KO VSMCs was not
significantly higher than with WT VSMCs (10.16% 2.96% SEM) (FIG. SA),
suggesting the lack of an NK cell activation signal on hPSC-derived VSMCs.
However, in agreement with the hypothesis, it was found that the percentage of
CD107a+ degranulating NK cells in a co-culture with KI-PHC VSMCs (5.43%
0.95% SEM) was significantly lower than in the presence of KO VSMCs (13.51%
2.51% SEM) (FIG. SA), suggesting that NK cell activity is indeed inhibited by
HLA-
G expression in KI-PHC VSMCs. FACS plots of one representative donor are shown
in FIG. 11C. The LDH 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).
Finally, macrophage activity was examined using a pH-sensitive fluorescent
dye (pHrodo-Red) that emits a signal upon phagocytic engulfment. It was
hypothesized that overexpression of the macrophage 'don't-eat-me' signal CD47
in
derivatives of the engineered hPSC cell lines would reduce macrophage
engulfment.
Given that no significant increase of CD47 expression was observed in KI-PHC
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). In
addition, 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
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donors. The emergence of red signal, an indicator of VSMCs that were engulfed
by
macrophages, was monitored by live cell imaging and the fluorescence intensity
was
quantified. Of note, with or without STS treatment, KI-PC VSMCs showed
significantly decreased engulfment by macrophages when compared to CD47-/- or
WT VSMCs (FIGS. 5C-5D, and FIG. 11E). These data demonstrate that
overexpression of CD47 can indeed minimize macrophage engulfment of engineered
hPSC-derived VSMCs, although a contribution of PD-Li to inhibiting macrophage
engulfment cannot be ruled out, which was also expressed by KI-PC VSMCs (FIG.
7F).
Discussion
In this study, multiplex CRISPR/Cas9 genome editing was applied to knock
out the highly polymorphic HLA-A/-B/-C genes, and successfully prevented the
expression of HLA class II genes by targeting the CIITA gene in hPSCs. In
addition,
CRIPSR/Cas9-assisted homology directed repair (HDR) was used to introduce the
immunomodulatory factors PD-L1, HLA-G and CD47 into the AAVS1 locus. It was
found that the engineered hPSC derivatives elicited significantly less immune
activation and killing by T cells and NK cells and displayed minimal
engulfment by
macrophages.
In the approach for ablating HLA class Ia expression, the polymorphic HLA
class Ia genes, HLA-A/-B/-C were specifically excised, while leaving the genes
B2M
and the nonpolymorphic HLA class lb genes HLA-E, -F 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. Interestingly, for unknown reasons, 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 al., 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. Out of
648
predicted off-target sites for the eight sgRNAs used, only one exonic off-
target event
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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. Even though
mutations
in the HLA-H have been linked to hereditary hemochromatosis, a rare iron
storage
disorder (Feder et al., 1996), the observed HLA-H (HFE) mutation did not
impact the
growth rate or differentiation efficiencies of the cell types tested in this
study. Of note,
it would be possible to avoid this off-target event, either by designing a
different
sgRNA or by selecting clones that do not harbor this particular off-target
mutation by
genotyping the HLA-H locus. Moreover, using sgRNA/Cas9 ribonucleoprotein
complexes (RNP) for targeting, which allows for more transient editing than
plasmid-
based approaches (Roth et al., 2018), should reduce the number of off-target
events
per cell line and thus be applied in the future.
As expected, the removal of polymorphic HLA expression in hPSCs and their
derivatives, such as ECs and VSMCs, resulted in reduced T cell responses in
vitro and
in vivo. An interesting observation from the T cell assays is that
overexpression of the
checkpoint inhibitor PD-Li only had a significant impact on the proliferation
and
cytotoxicity of CD8+ T cells. This may have several possible explanations: 1)
the
levels of the PD-Li receptor, PD-1, are higher on CD8+ T cells than on CD4+ T
cells.
2) CD8+ T cells are the cell type most responsive to target cell exposure in
the assays
and hence will also express higher levels of the negative regulator PD-1. Of
note,
both explanations are not mutually exclusive, as the strength of T cell
activation and
PD-1 expression are linked by a negative feedback loop (Riley, 2009).
Moreover, it
was noted that PD-Li alone had an impact on CD8+ T cell proliferation even in
the
absence of HLA, suggesting that PD-Li can act as a tolerogenic factor even in
the
absence of a productive HLA-TCR interaction. In addition, in the T cell
activation
and cytokine secretion assays, when compared to the negative control,
background T
cell activity was observed even in a co-culture with the KIPHC cell line. This
could
be due to the experimental setup, considering target cells may secrete factors
that
promote T cell activation independent of the presence of HLA.
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
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assays suggest that HLA-G expression was able to control NK cell activities.
Moreover, overexpression of CD47 effectively reduced macrophage engulfment.
Yet,
as recently reported, 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. With regards to long-term engraftment, in particular,
antibody-
dependent cellular cytotoxicity (ADCC) by NK cells, and allo-antibody-mediated
complement activation as the main drivers of chronic graft rejection must be
considered (Baldwin et al., 2016; Djamali et al., 2014; Michaels et al.,
2003). It can be
envisioned that introducing additional factors known to inhibit ADCC and
complement activation, such as CD59 (Men i et al., 1990), may enable durable
engraftment. Ultimately, in vivo experiments will help clarify the extent of
protection
that modified cells may have following transplantation. Yet, while various
humanized
mouse models exist, they are limited in re-capitulating a full human immune
response.
Thus, the development of improved in vivo models for testing cell
transplantation and
rejection may be required (Brehm et al., 2014; Li et al., 2018; Melkus et al.,
2006;
Rongvaux et al., 2014).
Overcoming the immune barrier to transplantation would provide an exciting
new modality not only to overcome the allobarrier, but also potentially to
treat
autoimmune diseases such as type 1 diabetes (TID) and multiple sclerosis,
where one
particular cell type is attacked by the patient's own immune system and needs
replacement. Thus, the generation of universal cells that can be safely
transplanted
into anyone holds the promise of unlocking the full potential of regenerative
medicine.
Experimental Procedures
CRISPR gRNA Sequences
HLA-A upstream: 5'-GCCGCCTCCCACTTGCGCT-3' (SEQ ID NO: 1)
HLA-A downstream: 5'-CACATGCAGCCCACGAGCCG-3' (SEQ ID NO: 2)
HLA-B upstream_l: 5'-ATCCCTAAATATGGTGTCCC-3' (SEQ ID NO: 3)
HLA-B upstream_2: 5'-TCCCTAAATATGGTGTCCCT-3' (SEQ ID NO: 4)
HLA-C downstream_l: 5'-GTGATCCGGGTATGGGCAGT-3' (SEQ ID NO: 5)
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HLA-C downstream_2: 5'-TGATCCGGGTATGGGCAGTG-3' (SEQ ID NO: 6)
CIITA: 5' -TCCATCTGGTCATAGAAG-3' (SEQ ID NO: 7)
gRNA_AAVS1-T2: 5'-GGGGCCACTAGGGACAGGAT-3' (SEQ ID NO: 8)
PCR and qPCR Probes/Primers
PCR primers used in FIG. 6:
Purple_F: 5'-CACTCAGAGCAAAGGTCAGATG-3' (SEQ ID NO: 9)
Purple_R: 5'-AGACTTGAATCCATAAGCCCAA-3' (SEQ ID NO: 10)
Red_F: 5'-GACAAGTCTCGGAGATGGTTTT-3' (SEQ ID NO: 11)
.. Red_R: 5'-AGACTTGAATCCATAAGCCCAA-3' (SEQ ID NO: 12)
Green_F: 5'-CACTCAGAGCAAAGGTCAGATG-3' (SEQ ID NO: 13)
Green_R: 5' -TTTGTTGTCAGCCAGACATAGG-3' (SEQ ID NO: 14)
Yellow_F: 5' -CTGGTTATCTCCCCATTCTCTG-3' (SEQ ID NO: 15)
Yellow_R: 5'-AAGCATTCACTCCTGACCCTG -3' (SEQ ID NO: 16)
Blue_F: 5'-GTCTTCCCTCCCAGGCAGCTCA-3' (SEQ ID NO: 17)
Blue_R: 5'-TGAGGGGTGGGGGATACCGGA-3' (SEQ ID NO: 18)
Black_F: 5' -TCGACCTACTCTCTTCCGCA-3' (SEQ ID NO: 19)
Black_R: 5'-TAGGGGGCGTACTTGGCATA-3' (SEQ ID NO: 20)
Gray_F: 5'-CCGTTCTCCTGTGGATTCGG-3' (SEQ ID NO: 21)
Gray_R: 5'-TCTCTGGCTCCATCGTAAGC-3' (SEQ ID NO: 22)
PCR primers used in FIG. 8:
HLA-F-ASl_F: 5'-GTCGCTTCAGTCAGGACACA-3' (SEQ ID NO: 23)
HLA-F-ASl_R: 5'-GAAGGTGCTGTTTGGCACAG-3' (SEQ ID NO: 24)
ITGA6_F: 5'-CCTTCAACTTGGACACTCGGG-3' (SEQ ID NO: 25)
.. ITGA6_R: 5'-CCACGGGCCAACTACTCC-3' (SEQ ID NO: 26)
HEATRl_F: 5'-TTACCCAGTTCAATACTGAGCCA-3' (SEQ ID NO: 27)
HEATRl_R: 5'-AGGGGTAAGCTGCAAACTTCTT-3' (SEQ ID NO: 28)
PTDSS2_F: 5'-GACCTCCACAGGGACTAGGT-3' (SEQ ID NO: 29)
PTDSS2_R: 5'-TTTGGAGTTGGTGCTCCCTC-3' (SEQ ID NO: 30)
CTBS_F: 5' -GCCCTCATCGAGTGGTCAAA-3' (SEQ ID NO: 31)
CTBS_R: 5'-CCGCTAGACCTGCTGCTATG-3' (SEQ ID NO: 32)
ACSBGLF: 5'-CTGGGTGTCAATGATGGCGT-3' (SEQ ID NO: 33)
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ACSBGLR: 5'-GCCACATCTAAAGGCAGTCG-3' (SEQ ID NO: 34)
AC078852.1_F: 5'-GTTTGTGGGTGCTGGTCAAC-3' (SEQ ID NO: 35)
AC078852.1_R: 5'-CTAGGCAACAGTGACAGGGG-3' (SEQ ID NO: 36)
HIPK4_F: 5'-GGACCATCATGTCGGAGACC-3' (SEQ ID NO: 37)
HIPK4_R: 5'-GACCTGGGAGTCACACGAAC-3' (SEQ ID NO: 38)
ACSBGLF: 5'-CTGGGTGTCAATGATGGCGT-3' (SEQ ID NO: 39)
ACSBGLR: 5'-GCCACATCTAAAGGCAGTCG-3' (SEQ ID NO: 40)
HIC2_F: 5'-AAGTGTTCGGTCTGCGAGAA-3' (SEQ ID NO: 41)
HIC2_R: 5' -GCTCTGCTTGGTACGGACTG-3' (SEQ ID NO: 42)
HLA-H_F: 5'-AGGTGATGTATGGCTGCGAC-3' (SEQ ID NO: 43)
HLA-H_R: 5'-TCCTTCCCGTTCTCCAGGTA-3' (SEQ ID NO: 44)
HLA-K_F: 5'-GGTATGAACAGCACGCCAAC-3' (SEQ ID NO: 45)
HLA-K_R: 5'-GCGTCTTGTGTTCCCTGGTA-3' (SEQ ID NO: 46)
HLA-G_F: 5'-ACCCTCTACCTGGGAGAACC-3' (SEQ ID NO: 47)
HLA-G_R: 5'-AGGCTCTCCTTTGTTCAGCC-3' (SEQ ID NO: 48)
PYCRL_F: 5'-CCTAGCCACGTGTGACTCAA-3' (SEQ ID NO: 49)
PYCRL_R: 5' -TGCCGTCCCAGTAACCAATC-3' (SEQ ID NO: 50)
RAB11FIP4_F: 5'-CGAGGGAGGGCAAATTGAGT-3' (SEQ ID NO: 51)
RAB11FIP4_R: 5'-GAAGAAGGGACAAGGGGTGG-3' (SEQ ID NO: 52)
CHFR_F: 5'-GAGCTTTGATGGCAGAGTGTTA-3' (SEQ ID NO: 53)
CHFR_R: 5'-CTGGGAGCATGCATTTGTGAGA-3' (SEQ ID NO: 54)
PNCK_F: 5'-CTGTTGGCAGGTGAACCTCT-3' (SEQ ID NO: 55)
PNCK_R: 5'-CTGGGAAGGCTTGTCTCCTG-3' (SEQ ID NO: 56)
AMN_F: 5'-AGAGCTCAAGGTCCCAAGTG-3' (SEQ ID NO: 57)
AMN_R: 5'-GGGTAACTCACTCGGAGGTC-3' (SEQ ID NO: 58)
FUTl_F: 5'-TGGATTTCCAGAACCCCATCC-3' (SEQ ID NO: 59)
FUTl_R: 5'-GGGAACTCTCCCTCTGGTCT-3' (SEQ ID NO: 60)
NPPA_F: 5' -GAGCTTCTGCATTGGTCCCT-3' (SEQ ID NO: 61)
NPPA_R: 5' -TCTGATCGATCTGCCCTCCT-3' (SEQ ID NO: 62)
SYBR-based qPCR primers:
AFP_F: 5' -AAATGCGTTTCTCGTTGCTT-3' (SEQ ID NO: 63)
AFP_R: 5' -GCCACAGGCCAATAGTTTGT-3' (SEQ ID NO: 64)
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SOX17_F: 5'-CTCTGCCTCCTCCACGAA-3' (SEQ ID NO: 65)
50X17_R: 5'-CAGAATCCAGACCTGCACAA-3' (SEQ ID NO: 66)
BRACHYURY_F: 5'-AATTGGTCCAGCCTTGGAAT-3' (SEQ ID NO: 67)
BRACHYURY_R: 5'-CGTTGCTCACAGACCACA-3' (SEQ ID NO: 68)
FLKl_F: 5'-TGATCGGAAATGACACTGGA-3' (SEQ ID NO: 69)
FLKl_R: 5'-CACGACTCCATGTTGGTCAC-3' (SEQ ID NO: 70)
MAP2_F: 5'-CAGGTGGCGGACGTGTGAAAATTGAGAGTG-3' (SEQ ID NO:
71)
MAP2_R: 5'-CACGCTGGATCTGCCTGGGGACTGTG-3' (SEQ ID NO: 72)
PAX6_F: 5'-GTCCATCTTTGCTTGGGAAA-3' (SEQ ID NO: 73)
PAX6_R: 5'-TAGCCAGGTTGCGAAGAACT-3' (SEQ ID NO: 74)
TaqMan gene expression assays:
HLA-E: Hs03045171_ml
CD8: Hs00233520_ml
IL-2: Hs00174114_ml
RPLPO (internal control): Hs99999902_ml
FAGS antibodies
a-HLA-A2 (PE-conjugated), Clone BB7.2, Biolegend, Cat#343305
a-HLA-ABC (PE-conjugated), Clone W6/32, Biolegend, Cat#311406
a-HLA-E (PE-conjugated), Clone 3D12, Biolegend, Cat#342603
a-HLA-G (PE-conjugated), Clone MEM-G/9, Abcam, Cat#ab24384
a-HLA-DR (APC-conjugated), Clone MEM-12, ThermoFisher Scientific, Cat#MA1-
10347
a-B2M (APC-conjugated), Clone 2M2, Biolegend Cat#316311
a-PD-Li (APC-conjugated), Clone 29E.2A3, Biolegend, Cat#329708
a-PD-1 (APC-conjugated), Clone EH12.2H7, Biolegend, Cat #329908
a-CD3 (APC-conjugated), Clone UCHT1, Biolegend, Cat#300412
a-CD3 (Pacific BlueTM-conjugated), Clone UCHT1, Biolegend, Cat#300418
a-CD4 (PE/Cy7-conjugated), Clone RPA-T4, Biolegend, Cat#300511
a-CD8 (PE-conjugated), Clone SK1, Biolegend, Cat#344705
a-CD25 (Alexa Fluor 700-conjugated), Clone M-A251, Biolegend, Cat#356117
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a-CD47 (PE-conjugated), Clone CC2C6, Biolegend, Cat#323108
a-CD56 (PE-conjugated), Clone HCD56, Biolegend, Cat#318306
a-CD69 (Alexa Fluor 647-conjugated), Clone FN50, Biolegend, Cat#310918
a-CD107a (APC-conjugated), Clone H4A3, Biolegend, Cat#328620
a-CD144 (PE-conjugated), Clone 55-7H1, BD Biosciences, Cat#560410
Isotypes
Isotype 1: Mouse IgG2b, K Isotype Control (APC-conjugated), Biolegend,
Cat#400322
Isotype 2: Mouse IgG2b, K Isotype Control (PE-conjugated), Biolegend,
Cat#401208
Isotype 3: Mouse IgG2a, K Isotype Control (PE-conjugated), Biolegend,
Cat#400214
Immunofluorescence antibodies
a-OCT4, Abcam, Cat#ab19857
a-NANOG, Abcam Cat#ab21624
a-SSEA3, Millipore, Cat#MAB4303
a-SSEA4, Millipore, Cat#MAB4304
a-TRA-1-60, Millipore, Cat#MAB4360
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate,
Life Technologies, Cat#A-21206
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate,
Life Technologies, Cat#A-21202
Goat anti-Mouse IgM Heavy Chain Secondary Antibody, Alexa Fluor 555
conjugate, Life Technologies, Cat#A-21426
Human ES Cell Culture, Electroporation, and Drug Selection
HUES8 cells (Cowan et al., 2004) were grown on Geltrex (Life Technologies)
pre-coated plates and cultured in mTeSR1 (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
electroporation, as previously described (Peters et al., 2013), HUES8 cells
were
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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. For gene knock-in into the AAVS1 locus, cells were electroporated
with
50 pg of pCas9_GFP, 25 pg of gRNA_AAVS1-T2 (Addgene #41818), and 40 pg of
double-stranded donor plasmid. For gene knock-out purpose, the cells were
collected
48 hrs post-electroporation. GFP-expressing cells were enriched by FACS
(FACSAria
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.
Alternatively, for gene knock-in, 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.
CRISPR/Cas9 Genome Editing
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 al., 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.n1) for on-target
efficiency.
Single guide RNAs with the highest on-target activities were used for genome
editing
in HUES8 cells. To build the knock-in donor plasmid, the ORFs of PD-L1, HLA-G,
and CD47 were individually cloned and connected by 2A sequence using Gibson
Assembly (New England BioLabs). The 3-in-1 cassette was then inserted into
the
AAVS1-Blasticidine-CAG-Flpe-ERT2 plasmid (Addgene #68461) between Sal I and
Mlu I restriction sites, after Flpe-ERT2 was cut out. Details on genome
editing of
human ESCs were previously described (Peters et al., 2013).
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Generation of HLA Knockout (KO) Cell Line
Briefly, to knockout the adjacent HLA-B/-C genes, a total of four sgRNAs was
co-electroporated together with a Cas9 expression plasmid into wild-type (WT)
HUES8. Primers shown in FIG. 6A were then used to screen for homozygous
knockout clones (HLA-B/-C-/- efficiency: 1.56%). Heterozygous knockout clones
were also observed (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).
At last, one karyotypically normal clone was chosen for further targeting of
the HLA-
A and CIITA genes in one electroporation. PCR with the primers shown in FIG.
6B
and flow cytometry using an a-HLA-A2 antibody were carried out to screen for
HLA-
A knockout clones. Primers shown in FIG. 6E and Sanger sequencing were
performed
to identify CIITA knockout clones. As a result, only heterozygous clones (HLA-
A'
CIITA') were observed after the first round of HLAA/CIITA targeting (HLA-A+/-
efficiency: 3.68%). Therefore, another round of electroporation with HLA-
A/CIITA
sgRNAs was applied to one karyotypically normal heterozygous clone, and the
same
screening strategies were employed. At last, one homozygous clone (HLA-A-/-
CIITAindelhndel) was generated, however, FACS analysis revealed that this
clone was
an admixed clone, which still retained 1% HLA-A cells. After subcloning, a
pure
homozygous clone (HLA Knockout, KO) was obtained.
Karyotyping
Karyotype G-banding was performed by Cell Line Genetics.
Directed Differentiation into Three Germ Layers
WT and gene-edited HuES8 cell lines were differentiated into ectoderm,
mesoderm, and endoderm following the monolayer-based protocols of the
STEMdiffTm Trilineage Differentiation Kit (StemCell Technologies).
Differentiation into Endothelial Cells and Vascular Smooth Muscle Cells
Human endothelial cells (EC) and vascular smooth muscle cells (VSMC) were
differentiated following the published protocols (Patsch et al., 2015).
Briefly, for EC
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differentiation, ESCs were plated in N2B27 media supplemented with 8 uM
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 pM forskolin (Abcam) 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
differentiation for at least 7 days. For VSMC differentiation, ESCs were
plated in the
same media for 3 days as for EC differentiation. On day 4 and 5, media were
changed
to N2B27 supplemented with 12.5 ng/ml PDGF-BB (Peprotech) and 12.5 ng/ml
Activin A (Cell Guidance Systems). From day 6 onwards, cells were dissociated
and
plated on gelatin-coated dishes in Medium 231 supplemented with Smooth Muscle
Growth Supplement (ThermoFisher Scientific) for further differentiation.
Human Primary Immune Cell Isolation and Culture
Blood was obtained from healthy, de-identified donors (leukopaks) from the
Jackson Transfusion Center at Massachusetts General Hospital, Boston. Human
primary T cells, NK cells, or CD14+ monocytes were isolated by negative
selection
kits (RosetteSepTM Human T Cell Enrichment Cocktail, RosetteSepTM Human NK
Cell Enrichment Cocktail, and RosetteSepTM Human Monocyte Enrichment Cocktail,
StemCell Technologies), respectively. Isolated T cells were cultured in X-VIVO
10
(Lonza) media supplemented with 5% Human AB Serum (Valley Biomedical), 5%
Fetal Bovine Serum, 1% Penicillin/Streptomycin, GlutaMAX, MEM Non-Essential
Amino Acids (ThermoFisher Scientific), and 20 U/ml IL-2 (Peprotech). Isolated
NK
cells were cultured in RPMI 1640 with L-Glutamine (Corning) supplemented with
10% Fetal Bovine Serum 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
PBS containing 1% Fetal Bovine Serum (FBS) was used as washing and
staining buffer; PBS containing 4% FBS was used as blocking buffer. In the
case of
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ECs and VSMCs, FcR blocking reagent (Miltenyi Biotec) was added to the
blocking
buffer at a 1:1000 dilution. Briefly, immune cells or other dissociated single
cells
were washed once and blocked with blocking buffer on ice for 20 min. Cells
were
stained with antibodies on ice for 30-60 mm and washed twice before analysis
on a
FACSCaliburTM or LSR II (BD Biosciences). The data were plotted using FlowJo
software (BD).
In Vitro T Cell Proliferation Assay
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 IL-2 for 5 days. 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.
In Vitro T Cell Activation Assay and Cytokine Secretion Assay
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.
After 5-day-co-culture, T cells were stained for T cell activation markers
before being
analyzed on an LSR II. For multiple secreted cytokine quantifications,
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. T cells activated with DynabeadsTM Human T-
Activator CD3/CD28 beads (ThermoFisher Scientific) for 5 days served as
positive
control. Background activation was assessed using T cells incubated with
conditioned
media from ECs or VSMCs. Conditioned media was prepared as described above.
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In Vitro T/NK Cell Killing Assay
ESC-derived VSMCs were used as target cells. For T cell killing assay, the
conditions for co-incubation were the same as in the T cell activation assay.
For NK
cell killing assay, 40K VSMCs and NK cells at the indicated effector/target
ratios
were co-incubated in 200 pl 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 LDH Cytotoxicity Assay Kit (ThermoFisher Scientific)
following the manufacturer's instructions. T cell medium or NK cell medium
(RPMI-
10) was used as background control. T/NK cells cultured alone or target cells
cultured
alone were used as controls for spontaneous LDH release. Lysed target cells at
endpoint were used as maximum LDH release.
Pre-sensitization of Allogeneic Human CD8+ T Cells
Human primary CD8+ T cells were isolated using RosetteSepTM Human CD8+
T Cell Enrichment Cocktail (StemCell Technologies), and pre-sensitized with
HUES 8-derived embryoid bodies as previously described (Gomalusse et al.,
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.
In Vivo T Cell Recall Response Assay
All animal experiments were performed in accordance to Harvard University
International Animal Care and Use Committee regulations. No randomization was
used. All procedures were done in a blinded fashion. Male immunodeficient SCID
Beige mice (Taconic) aged 8-10 weeks were used for teratoma formation. Two
million HUES8 cells were encapsulated in a blood clot, and the blood clot was
inserted subcutaneously into each flank of 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. Following T cell injection,
teratoma size was
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measured on day 2, day 5, and day 7; teratoma size was also measured 2 days
before
the T cell injection. On day 8 post-injection, the teratoma were harvested and
analyzed by qPCR and hematoxylin and eosin (H&E) staining. Two allogeneic CD8+
T cell donors were used in the same experimental condition and the results
were
combined in this study. Histology was performed by the histology core of the
Harvard
Stem Cell Institute.
In Vitro NK Cell Dc granulation Assay
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 mm 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.
NK cell cultures without target cells were used as negative control. NK cells
treated
with Cell Activation Cocktail (without Brefeldin A), which includes PMA
(phorbol
12-myristate-13-acetate) and ionomycin, were used as positive control for
degranulation.
In Vitro Macrophage Phagocytosis Assay
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 pt-plates (ibidi) at a density of 100K/well two days
before the
assay. For the assay, differentiated VSMCs were pretreated with 200 nM
staurosporine (Sigma) for 1.5 hrs to be used for the "STS treated" group.
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
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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 mm 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.
Generation of CD47-/- and B2M-/- HUES8 cell lines
The following four CRISPR sgRNAs were used to target the first coding exon
of CD47 in HUES8 cells.
5'-gGTCCTGCCTGTAACGGCGG-3' (SEQ ID NO: 75)
5' -gGACCGCCGCCGCGCGTCAC-3' (SEQ ID NO: 76)
5'-gCAGCAACAGCGCCGCTACC-3' (SEQ ID NO: 77)
5'-gTTCGCCCCCGCGGGCGTGT-3' (SEQ ID NO: 78)
Cells were stained 72 hrs post electroporation with an anti-CD47 antibody
(Clone CC2C6), and CD47 negative cells were isolated using a FACS Aria (BD).
Single cell-derived colonies were obtained as described previously (Peters et
al.,
2013), and subsequently loss of CD47 expression was confirmed by FACS
analysis.
Similarly, a Beta-2-Microglubulin (B2M)-deficient HUES8 cell line was
generated
using the following sgRNA:
5' -gCTACTCTCTCTTTCTGGCC-3'. (SEQ ID NO: 79)
Lentiviral Transduction
A doxycycline-inducible lentiviral Gateway vector (Invitrogen) containing the
.. PD-Li ORF was constructed by PCR amplification. PD-Li expression
lentiviruses
were packaged by transfecting HEK293T cells with the PD-Li -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. After
24 hrs, VSMCs were treated with doxycycline (10 pg/ml) to induce PD-Li
expression
that was verified by FACS. Assessment of T cell proliferation against VSMCs
overexpressing PD-Li was performed as described above, except for a 7-day-co-
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incubation and the presence of doxycycline throughout the co-incubation. No
effect
on T cell proliferation was observed by the addition of doxycycline.
Immunofluorescence
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%
paraformaldehyde, and permeablized with 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.
RNA Isolation, cDNA Synthesis and qPCR
RNA was extracted using TRIzol Reagent (ThermoFisher Scientific)
according to the manufacturer's instructions. cDNA synthesis was done using
SuperScript VILO cDNA synthesis kit (ThermoFisher Scientific) according to the
manufacturer's protocol. SYBR green-based or TaqMan-based qPCR was performed,
and relative quantification was determined using the QuantStudio 12k Flex
System
(ThermoFisher Scientific) and then calculated by means of the comparative Ct
method (2-AAct) relative to the expression of the respective internal control.
Next Generation Sequencing (NGS)-Based Off-target Analysis
The off-target sites were predicted using CCTop (Stemmer et al., 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.
Following genomic DNA extraction from WT as well as from the three engineered
hPSC lines, the biotinylated RNA baits were hybridized to the corresponding
denatured genomic DNA library. Subsequently, the RNA-gDNA hybrids were bound
to streptavidin-coated beads and non-specific bonds were washed off. The
remaining
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gDNA libraries were amplified and sequenced by paired-end NGS using NovaSeq
(IIlumina).
Genome editing events were quantified by CRISPRessoPooled from
CRISPResso suite (Version 1Ø13) with default settings unless stated later
(Pinello et
al., 2016). In brief, for each of the four libraries, the reads with minimum
single base
pair score (phred33) greater than 25 were selected and aligned to a 100bp
window
around each gRNA off-target site in the human genome (hg38). The sites (=3)
with
fewer than 5 aligned reads in any of the libraries were filtered out. The
percentage of
reads with altered sequences (insertion, deletion, and substitution) compared
to hg38
at each off-target site from each library was calculated by the program. If
the % 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.
Statistical Analyses
Plots were generated, and statistical analyses were performed using Prism 7
(Graphpad).
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