Editorial
Getting Into the Rhythm With CRISPR
Thierry VandenDriessche, Marinee K. Chuah
I nherited cardiac arrhythmias are a major health issue of
unmet medical need that can cause significant morbidity
and mortality. If left untreated, it represents a major cause of
sudden death in childhood. Available treatments include β-
blockers and implantable defibrillators, but these are only par-
tially effective and can still provoke significant adverse events,
including death. Catecholaminergic polymorphic ventricular
tachycardia is an autosomally transmitted form of inherited
cardiac arrhythmia that is caused in the majority of the cases
by gain-of-function missense mutations in the gene encod-
ing the RYR2 (ryanodine receptor 2). Such mutations in the
corresponding RYR2 gene result in mutant subunits that can
be incorporated into multimeric RYR2 receptors compromis-
ing their function. Consequently, these mutant dysfunctional
RYR2 receptors promote uncontrolled calcium leakage from
the sarcoplasmic reticulum triggering potentially lethal ar-
rhythmia. To increase the proportion of fully functional wild-
type RYR2 channels, the mutant RYR2 subunits should be
removed from the multimeric complexes. To selectively in-
hibit expression of these RYR2 mutant subunits gene therapy
could be used as an alternative solution to treat these types of
dominantly inherited cardiac arrhythmias that may overcome
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the limitations of the current standard of care.
Article, see p 953
Adeno-associated viral vectors (AAV) are well suited
to deliver therapeutic genes into the myocardium, particu-
larly those based on the AAV1 and AAV9 serotypes that
show increased cardiotropism.1 Initial preclinical studies in a
mouse model of catecholaminergic polymorphic ventricular
tachycardia had shown that this could be achieved by gene
therapy using AAV9 vectors expressing a short-hairpin (sh)
RNA specifically designed to selectively silence the mutant
Ryr2 gene by RNA interference.2 Consequently, this resulted
in a relatively reduction in total RyR2 protein levels and an
The opinions expressed in this article are not necessarily those of the
editors or of the American Heart Association.
From the Department of Gene Therapy and Regenerative Medicine,
Vrije Universiteit Brussel (VUB), Belgium (T.V., M.K.C.); Center for
Molecular and Vascular Biology, Department of Cardiovascular Sciences,
University of Leuven, Belgium (T.V., M.K.C.).
Correspondence to Thierry VandenDriessche, MSc, PhD, Department
of Gene Therapy and Regenerative Medicine, Vrije Universiteit Brussel
(VUB), Bldg D, Room D365, Laarbeeklaan 103, B-1090 Brussels,
Belgium, Email thierry.vandendriessche@vub.ac.be; or Marinee K.
Chuah, Department of Gene Therapy and Regenerative Medicine, Vrije
Universiteit Brussel (VUB), Bldg D, Room D365, Laarbeeklaan 103,
B-1090 Brussels, Belgium, Email marinee.chuah@vub.ac.be
(Circ Res. 2018;123:928-930.
DOI: 10.1161/CIRCRESAHA.118.313876.)
© 2018 American Heart Association, Inc.
Circulation Research is available at https://www.ahajournals.org/
journal/res
DOI: 10.1161/CIRCRESAHA.118.313876
928
improvement of adrenergically stimulated ventricular tachy-
cardia. Nevertheless, the overall efficacy of this shRNA-based
strategy was limited because only a net 15% reduction in
RyR2 expression could be achieved and challenges related to
specificity remained. Moreover, this strategy would require
sustained shRNA expression throughout the entire lifespan of
the affected patients, but it is currently not known how long
expression from AAV vectors will last.
To overcome some of these limitations, gene-editing ap-
proaches could be used instead that directly target the mutated
RyR2 gene itself at the DNA level. In this issue, Pan et al3
established proof of concept that selective inactivation of the
mutant Ryr2 gene could be achieved using the CRISPR (clus-
tered regularly interspersed short palindromic repeat)/Cas sys-
tem (Figure). The CRISPR/Cas system is a natural defense
system in prokaryotes that targets invading phages or plasmids
by sequence-specific DNA cleavage that has been adapted for
use in eukaryotic cells.4 The true merit of the CRISPR system
is that a single Cas9 endonuclease can be used in combination
with any guide (g)RNA of choice to induce a site-specific dou-
ble strand DNA break in the desired target genes that stimulate
gene repair by either nonhomologous end joining or homolo-
gous recombination in the presence of a homologous DNA
template. This obviates the need to design a separate nuclease
for each intended DNA target sequence, greatly enhancing
the versatility of this gene-editing platform. Nonhomologous
end joining can result in the insertion or deletion (ie, indels)
of nucleotides at the target locus, resulting in frame shift or
nonsense mutations leading to a loss-of-function phenotype.
Though gene editing using CRISPR/Cas9 has been particu-
larly effective to generate transgenic animals containing spe-
cific mutations in the desired target loci, in vivo gene editing
in postnatal animals has been more challenging. Nevertheless,
recent studies showed that liver,5,6 heart, and skeletal mus-
cle7–10 could be targeted with CRISPR/Cas9, even resulting in
sustained therapeutic benefits in murine disease models.11
These studies strongly suggest that CRISPR/Cas9 may be
particularly attractive to inactivate mutant alleles that cause
a dominant inherited disease, like catecholaminergic poly-
morphic ventricular tachycardia. Pan et al3 now generated an
all-in-one AAV9 vector that coexpressed the gRNA targeting
the mutant Ryr2 allele and a small Cas9 variant (derived from
Staphylococcus aureus)4 that could all fit within the same vec-
tor backbone. The AAV9-gRNA/saCas9 vector was injected in
10-day old mice heterozygous for the RyR2 mutation R176Q
that mimic catecholaminergic polymorphic ventricular tachy-
cardia. This resulted in a specific gene disruption of the
pathogenic Ryr2 allele encoding the R176Q mutant protein,
consistent with the presence of indels at the DNA cleavage
site targeting by the gRNA. Consequently, this resulted in a
reduction of the corresponding total Ryr2 mRNA and pro-
tein levels. Most importantly, this reduction was associated
 VandenDriessche and Chuah   Getting Into the Rhythm With CRISPR   929

Figure. CRISPR/Cas9-based gene editing of dominant inherited arrhythmia. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an
autosomally transmitted form of inherited cardiac arrhythmia that is caused by gain-of-function missense mutations in the gene encoding the RYR2 (ryanodine
receptor 2). Mutant dysfunctional RYR2 receptors promote uncontrolled calcium leakage from the sarcoplasmic reticulum into the cytoplasm triggering lethal
Downloaded from http://ahajournals.org by on October 25, 2018

arrhythmia. A heterozygous (R176Q/+) murine model of CPVT had been developed in which 1 Ryr2 allele corresponded to an R176Q gain-of-function mutation,
whereas the other allele was wild-type. To increase the proportion of fully functional wild-type RYR2 channels, the mutant RYR2 monomers were selectively
removed from the multimeric complexes by specific disruption of the dominant mutant Ryr2 allele using CRISPR/Cas9. The wild-type allele was unaffected,
hence favoring the incorporation of wild-type RyR2 monomers into the RYR2 multimeric receptor, at the expense of the mutant RyR2 monomers. The CRISPR/
Cas9 components were delivered to the CPVT Ryr2 R176Q/+ mice using an all-in-one adeno-associated viral 9 vector coexpressing the saCas9 along with a
guide RNA specifically designed to recognize the mutant Ryr2 allele. Consequently, this resulted in a relatively robust phenotypic correction consistent with
prevention of arrhythmia in accordance with the normalization of the corresponding ECG pattern. NHEJ indicates nonhomologous end joining.

with a restoration of normal calcium handling, protecting the
heterozygous R176Q/+ mice from pacing-induced cardiac
arrhythmias.
To our knowledge, this is the first study demonstrating rel-
atively robust phenotypic correction in vivo of inherited car-
diac arrhythmia using CRISPR/Cas. It is encouraging that the
targeting efficiency and overall therapeutic effect seem more
robust than what had been achieved recently based on RNA
interference.2 However, some caution is warranted because
no head-to-head comparisons were performed to compare the
relative efficacy of shRNA- versus CRISPR/Cas9-based tar-
geting. It is reassuring that Pan et al3 showed that the gene
inactivation was specific for the mutant Ryr2 allele, where-
as the wild-type allele was unaffected. This is because of
the gRNA that was specifically designed to target a specific
polymorphism upstream of the Ryr2 mutation that is absent
in the wild-type locus. Though this approach discriminates
effectively between wild-type and mutant alleles, it would
need to be specifically adapted and personalized for each pa-
tient individually. Ideally, an one-size-fits-all design may be
preferred taking advantage of the most commonly occurring
polymorphisms or gain-of-function mutations in the RyR2
gene. Furthermore, the targeting specificity was confirmed
by the lack of any detectable off-target effects in nontarget
genes, enhancing the overall safety of this gene-editing strat-
egy. Nevertheless, to rule out potential off-target effects, more
comprehensive and unbiased genome-wide assays.12This
overcomes the limitations of computational algorithms that
typically rely on a limited set of computationally predicted
off-target sites and were therefore not highly predictive.
Because Cas9 and the Ryr2-specific gRNA are driven
from robust constitutive promoters (ie, CAG and U6 promoter,
respectively), it is likely that their expression will be sustained
after AAV transduction in cardiomyocytes though this was
not tested directly in the Pan et al3 study. However, ideally,
transient expression of the CRISPR components may be pre-
ferred as a hit-and-run strategy6 to minimize not only the risk
of putative off-target DNA cleavage but also possible inadver-
tent immune reactions directed against the Cas9-expressing
cardiomyocytes. In principle, transient expression of the
CRISPR components should suffice to achieve a permanent
modification of the target DNA at the desired locus, obviating
the need for continuous expression as in the case of shRNA-
based approaches. Whether gene editing also occurs in non-
cardiac cells that express RyR2, such as smooth muscle cells
and neurons requires further studies. One potential caveat of
 930  Circulation Research  September 28, 2018
the experimental design of the study by Pan et al3 is that rela-
tively high vector doses were required ≥1014 vector genomes
per kg to obtain a therapeutic effect. Because the CRISPR/
Cas AAV vector was injected subcutaneously, it may not have
been the ideal route to achieve efficient cardiac transduction
and other intravascular routes or direct intramyocardial ad-
ministration may be preferred. Though such high vector doses
have also been used in gene therapy trials, it carries a greater
risk of inducing AAV vector-associated hepatic inflammation
and toxicity. Typically these adverse events are subclinical
and can be contained by transient immune suppression with
tapering doses of corticosteroids resulting in sustained thera-
peutic effects for several years after liver or muscle-directed
gene therapy.13,14 To further reduce the vector doses used in
gene therapy clinical trials vectors and minimize the risk of
hepatic inflammation, it may be desirable to further enhance
the overall cardiac transduction efficiency of AAV vectors by
genetically modifying the AAV capsids that could be used in
conjunction with improved catheter-based delivery methods.
Alternatively, more robust promoters could be used to drive
the CRISPR components to further enhance the overall target-
ing efficiency. Though several outstanding questions remain
to be addressed, the study of Pan et al3 strongly supports the
use of CRISPR/Cas for the treatment of inherited dominant
cardiac arrhythmias. It is likely that the rapid advances in the
gene-editing field will improve the prospects of ultimately
moving this technology towards clinic applications for heart
disease. This may have broad implications not just for Ryr2-
associated cardiac arrhythmias but also for other dominant
Downloaded from http://ahajournals.org by on October 25, 2018
hereditary heart diseases.15
Sources of Fundings
T. VandenDriessche and M.K. Chuah are supported by grants from the
Fonds voor Wetenschappelijk Onderzoek (FWO); Koning Boudewijn
Stichting—Walter Pyleman Fund and Generet Fund; Telethon
VUB/UZ Willy Gepts grant, VUB Strategic Research Program and
Industrieel Onderzoeksfonds, Téléthon Belgique ABMM.
Disclosures
None.
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Key Words: Editorials ◼ clustered regularly interspaced short palindromic
repeats ◼ ryanodine ◼ sarcoplasmic reticulum ◼ tachycardia