44 128
(Figure S4A), also observed in the original parental line and in all
daughter clones after post hoc analysis. The 1q32 amplification
is common in hPSCs after extended propagation in culture (De-
kel-Naftali et al., 2012), and thus was not a result of CRISPR-
mediated off-target activity. To determine off-target activity of
our gRNAs, the top ten homologous sites per guide were deter-
mined by COSMID (Cradick et al., 2014) and sequenced in all
clonal and parental lines. No off-target mutations were observed
at any site (Table S2). All variants, besides a heterozygous SNP in
chromosome 11, were detected in less than 1% of reads, which
is consistent with error in the sequencing method.
Dystrophin (DYS
D
45–55
) Expression Is Restored in
Reframed DMD hiPSC-Derived Cardiomyocytes and
Skeletal Myotubes
CRISPR/Cas9-mediated deletion of
DMD
should result in an
internally deleted dystrophin protein lacking exons 45 55 (here-
after referred to as DYS
D
45–55
). As hiPSCs do not express dystro-
phin, we differentiated the reframed DMD hiPSC clonal lines to
two disease-relevant cell types, cardiomyocytes and skeletal
muscle myotubes, using directed differentiation or overexpres-
sion (OE) of MyoD to evaluate rescue of DYS
D
45–55
. PCR and
sequencing of the exon 44/56 boundary in cDNA from the re-
framed cardiomyocyte clones demonstrated correct splicing of
the dystrophin transcript (Figures S4C and S4D). Additionally,
both the reframed cardiac and skeletal muscle cell lines restored
dystrophin expression as assayed by immunocytochemistry and
western blot (Figures 3A–3C). Compared to wild-type CDMD
1002 or human skeletal muscle myotubes (HSMM), the band
was truncated by 66 kDa as expected.
DYS
D
45–55
Protein Restores Membrane Functionality to
Cardiomyocytes and Skeletal Myotubes In Vitro
Cardiomyocytes or skeletal myotubes lacking dystrophin
demonstrate membrane fragility in vitro and respond to osmotic
stress by releasing elevated levels of CK (Guan et al., 2014;
Menke and Jockusch, 1995), as is seen in human patients
(Pearce et al., 1964). To determine whether DYS
D
45–55
could
restore stability to dystrophic plasma membranes, we subjected
differentiated cardiomyocytes and skeletal muscle myotubes
derived from reframed and out-of-frame hiPSCs to hypo-os-
motic conditions. Cells were stressed by incubation in hypo-
osmolar solutions (66–240 mosmol) and CK release into the
supernatant was measured to show functional improvement
after dystrophin restoration. Both the reframed CDMD 1003-49
cardiomyocytes and skeletal muscle cells demonstrated re-
duced CK release, similar to wild-type (CDMD 1002), versus
the out-of-frame CDMD 1003 cells, indicating that DYS
D
45–55
was capable of reducing membrane fragility (Figure 4A). The
same trend was also observed with CDMD 1006/1006-1 cardio-
myocytes (Figure S4E). After normalizing and pooling all experi-
ments, we observed that significantly less CK was released at
93, 135, and 240 mosmol in the reframed and wild-type cells
compared to out-of-frame (Figure S4F).
CRISPR/Cas9 Reframing Correlates with miR31 Levels
in Skeletal Myotubes In Vitro
Elevated levels of miR31 have been observed in DMD patient bi-
opsies compared to wild-type or BMD (Cacchiarelli et al., 2011).
We measured levels of miR31 using droplet digital PCR (ddPCR)
after differentiation of out-of-frame and reframed CDMD hiPSCs
to skeletal myotubes. Reframing
DMD
reduced levels of miR31
(similar to wild-type cells) compared to out-of-frame
DMD,
as
is observed in human dystrophinopathies (Figure 4B). Thus, re-
framing the
DMD
gene normalizes miR31 levels similar to
BMD, demonstrating functional rescue of the dystrophic pheno-
type to a BMD phenotype.
DYS
D
45–55
Protein Restores the DGC In Vitro and In Vivo
As a third assay of DYS
D
45–55
functionality, we evaluated its abil-
ity to restore the DGC in vitro and in vivo. The DGC member
b
-dystroglycan was restored and detected at the membrane of
reframed hiPSCs, but not out-of-frame hiPSCs, after directed
differentiation to skeletal muscle in vitro by immunostaining
and western blot (Figures 4C and 4D). Additionally, skeletal mus-
cle cells derived from a wild-type (CDMD 1002), out-of-frame
(CDMD 1003), or reframed (CDMD 1003-49) hiPSC line were in-
jected into the tibialis anterior (TA) of NOD
scid
IL2Rgamma
(NSG)-mdx mice. Correctly localized dystrophin and
b
-dystro-
glycan was only observed in engrafted human cells (demarked
by human lamin A/C and spectrin) from the reframed or wild-
type lines (Figures 4E and 4F). These studies taken together
with the hypo-osmotic stress assays demonstrate the ability of
DYS
D
45–55
to functionally reassemble the DGC and restore mem-
brane stability in vitro and in vivo.
DISCUSSION
Using CRISPR/Cas9 gene editing, we have induced the largest
deletion accomplished to date in DMD hiPSCs and restored a
functional dystrophin protein. Deletion of
DMD
exons 45–55
has the potential to be therapeutically relevant to 60% of DMD
patients. Since this internal deletion has been associated with
a very mild disease course in multiple independent patients, a
therapy utilizing this approach should create a highly functional
dystrophin. We showed successful deletion of exons 45–55
using a single gRNA pair and did not identify any off-target activ-
ity at the top ten homologous sites; however, a more compre-
hensive and unbiased approach should be undertaken such
as whole-genome sequencing. Importantly, removal of exons
45–55 resulted in stable dystrophin protein (DYS
D
45–55
) in both
cardiomyocytes and skeletal myotubes in vitro. Functionality of
deleted. Deletion PCR genotyping results for six hiPSC clonal lines is shown. One pair of primers (red arrows in A) was located internal to the deletion and only
produced a 1,201 bp band in the undeleted clones CDMD 1003-13 and 1003-51. Another primer set (purple arrows in A) flanked the deletion region and produced
a 788 bp band only when the deletion and NHEJ occurred successfully, as in the reframed clones CDMD 1006-1, 1003-49, 1003-57, and 1003-81.
(C) Each clonal line maintained normal morphology (brightfield) and expressed NANOG (green) and SOX2 (red) by immunocytochemistry. Scale bar, 100
m
m.
Shown to the right is the sequence of the gDNA at the rejoining site between introns 44 (I44) and 55 (I55). Sequencing revealed a 16 bp deletion in CDMD 1006-1, a
2 bp insertion in CDMD 1003-49, and 1 bp insertions in CDMD 1003-57 and CDMD 1003-81.
See also Figures S1, S2, S3, S4A, and S4B.
536
Cell Stem Cell
18
, 533–540, April 7, 2016
ª
2016 Elsevier Inc.