CRISPR and cardiovascular diseases.pdf

Cardiovascular Research (2023) 119, 79–93 INVITED REVIEW
https://doi.org/10.1093/cvr/cvac048
CRISPR and cardiovascular diseases
Kiran Musunuru 1,2
*
1
Cardiovascular Institute, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA; and 2
Department of Genetics, Perelman School of
Medicine at the University of Pennsylvania, Philadelphia, PA, USA
Received 8 November 2021; revised 13 February 2022; accepted 15 February 2022; online publish-ahead-of-print 7 April 2022
Abstract CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on
biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i)
the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epi
genome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem
cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, fo
cusing on the examples of hypercholesterolaemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.
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Keywords gene editing • genome editing • base editing • CRISPR • cardiovascular disease
* Corresponding author. Tel: +1 (215) 573 4717; fax: +1 (215) 746 7415, E-mail: kiranmusunuru@gmail.com
© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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1. Clinical vignette
A 65-year-old man has been diagnosed with transthyretin amyloidosis
after starting to experience shortness of breath while walking his dog,
despite being very physically active for all his life. His father had died
of the same disease at age 67, and upon genetic testing, he is found to
be heterozygous for a pathogenic variant in the TTR gene that encodes
the transthyretin (TTR) protein responsible for the disease. To treat the
amyloidosis affecting his heart and nerves, he receives an intravenous in
fusion that delivers CRISPR gene-editing machinery directly into the he
patocytes in his liver, which inactivates almost all the copies of the TTR
gene in the liver. Within weeks, the amount of transthyretin in his blood
has dropped by ≈90%, and he reports that his symptoms have signifi
cantly improved. He is hopeful that his symptoms have been definitively
addressed and that he will not succumb to the same disease to which he
lost his father.
Although this case might have the feel of science fiction, in fact, it has
already played out in real life,1
published in a report in June 2021 and
widely covered in the press. CRISPR editing to treat cardiovascular dis
eases has arrived, highlighting the tremendous progress that has taken
place in the field over the past decade. This review discusses the founda
tional CRISPR technologies (Table 1) and how they are impacting cardio
vascular basic science research and the development of novel therapies.
Because CRISPR is now so widely used by so many investigators, this re
view does not attempt to comprehensively summarize all its applications
to cardiovascular research. Rather, it focuses on general principles and
illustrates them with a limited number of instructive examples.
2. CRISPR technologies
2.1. Standard nuclease gene editing
The original gene-editing approach, first developed in the 1990s, entailed
the use of nuclease proteins that controllably introduce double-strand
breaks (DSBs) at desired target sites in the genome.2
DSBs instigate cel
lular DNA repair responses, the most frequent response being non-
homologous end-joining (NHEJ).3,4
In NHEJ, the free DNA ends created
by the DSBs are rejoined in an error-prone process that can occasionally
insert or delete DNA base pairs, i.e. indel mutations. Such indel muta
tions can disrupt the protein products encoded by target genes (i.e.
frameshift mutations, in-frame codon insertions or deletions, splice
site mutations) or disrupt non-coding regulatory elements that influence
gene expression (Figure 1). Although NHEJ can yield highly efficient mu
tagenesis, approaching 100% efficiency in some contexts, the induced in
del mutations are semi-random in nature, meaning that it is difficult to
predict exactly which mutations will emerge in edited cells; while they
are typically small (one or a few base pairs) they can range to very large
sizes (kilobases). Accordingly, the imprecise nature of NHEJ makes it
best suited to the knockout of genes or regulatory elements. An excep
tion is the use of two nucleases to make DSBs in the same chromosomal
region, whereby NHEJ will efficiently join the far DNA ends generated
by the DSBs and eliminate the intervening sequence in a reasonably pre
cise manner, providing an attractive means to delete defined portions of
genes, entire genes, or even multiple genes (Figure 1).
The second major DSB repair mode is homology-directed repair
(HDR).3,5
In contrast to NHEJ, HDR is largely limited to proliferating
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cells that are in the S phase or G2 phase and thus have double the usual
DNA content. HDR uses a homologous region of DNA—which can be
on the duplicate chromatid on the same chromosome, on a sister
chromosome, or on a heterologously introduced synthetic single-strand
oligonucleotide or double-strand DNA vector—as a repair template to
precisely replace the DNA around the site of the DSB. If the repair tem
plate carries an alteration flanked by homologous sequences, HDR can
stably incorporate the alteration into the genome (Figure 1). This phe
nomenon makes HDR best suited for the introduction or correction
of mutations, as well as the insertion of cassettes. Even in permissive
cells, HDR is typically much less efficient than NHEJ, and NHEJ can com
plicate efforts to yield cells with precise genotypes (e.g. HDR correcting
a heterozygous mutation on one allele but NHEJ knocking out the other
allele with an indel mutation).
Although there are a variety of programmable nucleases capable of ef
ficiently introducing DSBs at target genomic sites, by far the most widely
used are CRISPR–Cas systems.6
The prototypic system, Streptococcus
pyogenes CRISPR–Cas9 (SpCas9), is the most popular due to its ease
of use and its relatively high editing efficiencies. This system comprises
the Cas9 protein, which is a nuclease that cuts the two DNA strands
with two distinct catalytic domains and is often likened to molecular scis
sors, and a single guide RNA (gRNA) ≈100 nucleotides in length, which is
often likened to a GPS that directs Cas9 to the target genomic site.7
Cas9
tightly complexes with the gRNA and scans across double-strand DNA
molecules, pausing at protospacer-adjacent motifs (PAMs)—which in the
case of SpCas9 are NGG sequences (N = any base), occurring on aver
age at 1 out of 16 positions along any given DNA strand. If the first 20
nucleotides at the 5′
end of the gRNA (spacer sequence) match the 20
nucleotides on the same DNA strand as the PAM and just upstream of
the PAM (protospacer sequence, on the non-target DNA strand), the
spacer sequence will hybridize with the complementary sequence on
the other DNA strand (target strand) via Watson–Crick base pairing,
which activates Cas9 to make a blunt-end DSB three base pairs upstream
of the PAM. The Cas9/gRNA complex remains intact and can continue to
search for and cleave target sites—including the site of any accurately re
paired DSB that it had previously induced.
Besides SpCas9, there are a variety of other CRISPR–Cas9 systems
that have been adapted from various bacterial species, e.g.
Staphylococcus aureus CRISPR–Cas9 (SaCas9) which is often used due
to its smaller size compared with SpCas9.8
The various Cas9 proteins
have distinct PAM preferences, e.g. SaCas9 prefers NNGRRT sequences
(R = G or A). Protein engineering has yielded variants of SpCas9 and
SaCas9 with altered PAM preferences, including near-PAMless SpCas9
variants that have relaxed PAM requirements, greatly increasing the
range of genomic sites that can be targeted.9
A distinct Cas family, the
Cas12 proteins, are also available for use in standard nuclease gene
...........................................................................................................................................................................................
Table 1 Comparison of CRISPR technologies
Technology Types of intended
edits
Types of unintended edits Efficiency Size of gene
encoding the
editing protein
Components besides
editing protein
Standard nuclease gene
editing—
non-homologous
end-joining
Semi-random, small indel
mutations; precise
deletions if two guide
RNAs used
Small or large indel mutations;
loss of chromosomal
segments; chromosomal
rearrangements;
chromothripsis
(chromosome shattering)
Can be very high
(close to
100%
efficiency)
Medium (in some
cases can fit in
a single AAV
vector)
Guide RNA; two guide RNAs
for precise deletion
Standard nuclease gene
editing—
homology-directed
repair
Precise single-nucleotide
changes, insertions,
deletions, and any
combination thereof
Small or large indel mutations;
loss of chromosomal
segments; chromosomal
rearrangements;
chromothripsis
(chromosome shattering)
Typically low Medium (in some
cases can fit in
a single AAV
vector)
Guide RNA; single-strand or
double-strand DNA repair
template
(homology-directed
repair)
Base editing Single-nucleotide changes
(typically restricted to
transition mutations)
Small indel mutations; bystander
single-nucleotide changes at
target site; off-target DNA
deamination; off-target RNA
deamination
Can be very high
(close to
100%
efficiency)
Large (typically
requires
splitting across
multiple AAV
vectors)
Guide RNA
Prime editing Precise single-nucleotide
changes, insertions,
deletions, and any
combination thereof
Small indel mutations; off-target
prime edits; unknown
consequences of reverse
transcriptase activity
Typically low
(but
improving
with new
innovations)
Large (typically
requires
splitting across
multiple AAV
vectors)
Prime editing guide RNA
(pegRNA); two pegRNAs
for deletion or twin prime
editing
Epigenome editing No sequence changes;
changes to methylation
status and/or
chromatin
modifications
Off-target methylation and/or
chromatin changes; edits
might not be durable
Can be high Medium to large Guide RNA
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CRISPR and cardiovascular diseases 81
editing; the best characterized are Cas12a/Cpf1, Cas12b/C2c1, and
Cas12e/CasX.10–12
In contrast to Cas9 proteins, Cas12 proteins use a
single catalytic domain to cut both DNA strands in a staggered configur
ation, tend to have PAM preferences weighted towards T-rich se
quences, use protospacer DNA sequences downstream of the PAMs,
and use spacer sequences at the 3′
ends of the gRNAs.
Standard nuclease gene-editing carries non-trivial risks of genotoxi
city. Even if DSBs occur at the desired target genomic sites, they can re
sult in large insertions or deletions that affect multiple genes, loss of
entire chromosomal segments, chromosomal rearrangements, and
even chromothripsis (chromosome shattering).13
This on-target collat
eral damage is increasingly coming to light as newer long-read
sequencing technologies are used to interrogate the targeted loci.
DSBs occurring at sites other than the intended target sites can result
in off-target mutagenesis, which carries the theoretical risk of disrupting
tumour suppressor genes or activating oncogenes and increasing the po
tential for carcinogenesis. A wide variety of techniques are available to
assess CRISPR–Cas systems for their propensity for off-target mutagen
esis, and when it occurs, it tends to be at genomic sites with a high de
gree of sequence similarity to the intended target site (limited number of
mismatches or bulges between the spacer and protospacer se
quences).14
Off-target mutagenesis can be mitigated via either Cas pro
tein engineering or modification of the guide RNA, though often with an
accompanying reduction of on-target editing efficiency.15–18
Figure 1 Standard nuclease gene editing with CRISPR–Cas9. CRISPR–Cas9 recognizes a genomic site defined by complementary base pairing of ≈20
nucleotides in the guide RNA (spacer) with the target DNA strand and the presence of an appropriate PAM in the non-target DNA strand. The arrows
indicate the sites of Cas9-mediated DNA cleavage to generate a double-strand break. Different outcomes occur depending on how many guide RNAs are
used, whether a custom-made DNA repair template is provided, and whether the double-strand break is repaired by NHEJ or HDR. Downloaded
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2.2. Base editing
CRISPR–Cas9 lends itself to other modes of editing besides standard nu
clease gene editing because the DNA cleavage activity and the genomic tar
geting activity are separated into two molecules, the Cas9 protein, and the
gRNA, respectively. With partial or complete elimination of the cleavage
activity of Cas9—either by mutating one of the catalytic domains such
that Cas9 can only nick one DNA strand (nickase Cas9, or nCas9) or by
mutating both catalytic domains such that Cas9 cannot cut either DNA
strand (dead Cas9, or dCas9)—the Cas9/gRNA complex can still localize
to a desired target genomic site, allowing the complex to serve as a plat
form on which to tether additional enzymes at the target site.
In base editing, nCas9 is fused to either a cytidine deaminase domain
(cytosine base editing) or an adenosine deaminase domain (adenine base
editing).19,20
There are a wide variety of cytosine base editors, using
cytidine deaminase domains adapted from the many naturally occurring
proteins that act upon cytosine bases in single-strand DNA molecules.6
In contrast, there are relatively fewer adenine base editors; because
there are no naturally occurring deaminase proteins that act upon aden
ine bases in single-strand DNA molecules, these editors all rely on the
same group of adenosine deaminase domains that were evolved in the
laboratory from a single deaminase protein (TadA) that acts upon aden
ine bases in single-strand RNA molecules.20
Both cytosine and adenine base editors can catalyse site-specific,
single-nucleotide edits, and as such their action is often likened to a pencil
and eraser. When the nCas9/gRNA complex engages with the target
genomic site, the non-target DNA strand assumes a single-strand con
formation as the gRNA hybridizes with the target DNA strand (an
R-loop structure), which makes any bases within a certain window on
the non-target strand accessible to the deaminase domain fused to
Figure 2 Base editing. Tethering of a deaminase domain to nickase Cas9 (nCas9) can result in site-specific C-to-T or A-to-G edits on the non-target
strand without the need for double-strand breaks. The arrow indicates the site of a nick.
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nCas9 (Figure 2). Cytosine base editors convert cytidine (C) to uridine
(U), and adenine base editors convert adenosine (A) to inosine (I).
Being non-standard in DNA, U is normally restored to C by the action
of the repair enzyme uracil-DNA glycosylase; fusion of yet another do
main to nCas9, a protein inhibitor of uracil-DNA glycosylase, prevents
this restoration. (Although I is non-standard as well, restoration to A is
slow, and there is no need to block an endogenous repair pathway for
adenine base editing.) The nickase activity of nCas9 is directed to the tar
get DNA strand only, which instigates a nick repair process that removes
a patch of nucleotides from that strand. Those nucleotides are replaced
via polymerase action that uses the complementary bases on the non-
target strand as the template, inserting an A opposite any U (which is
treated like a T), or a C opposite any I (which is treated like a guanosine,
or G). After the base editor has moved away from the site and nick repair
is complete, any U or I in the non-target strand is eventually replaced
with T or G via base excision and replacement by complementarity to
the opposing A or C on the target strand—completing the C–G to T–
A, or A–T to G–C, base pair change. (In unusual circumstances, a C–G
base pair can be edited to G–C by a cytosine base editor.21
)
Base editors are limited in the types of edits they can make (largely
C→T and A→G transition mutations) and the locations in which they
can make the edits (within the specific window on the non-target strand
dictated by the choice of base editor/deaminase and the positioning of
the PAM). There is also the possibility of undesirable bystander edits,
i.e. editing of bases other than the target base within the editing window.
Nonetheless, if the desired edit—whether a nonsense mutation or
splice site mutation to knock out a gene, or the correction of a disease-
causing mutation—is amenable to the action of a base editor, the editing
Figure 3 Prime editing. Tethering of an RT domain to nickase Cas9 (nCas9) and addition of a 3′
extension to the guide RNA to add a primer-binding
sequence (PBS) and RTT allows for RT to generate a variant-bearing DNA flap attached to the nicked non-target strand, which can ultimately replace the
corresponding portion of the original sequence. The arrow indicates the site of a nick.
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can potentially occur with close to 100% efficiency even in non-
proliferating cells. In such cases, base editing combines the high efficiency
of NHEJ and the precision of HDR while mitigating the disadvantages of
these repair modes of standard nuclease gene editing. Still, off-target
editing remains a concern. Although largely limited to single-nucleotide
changes—with minimal risk of large deletions or chromosomal rearran
gements due to base editing not relying on DSBs—stochastic off-target
editing from the deaminase domain interacting with single-stranded
DNA regions in a gRNA-independent fashion has been observed, almost
exclusively with cytosine base editors.22,23
As with standard nuclease
gene editing, off-target base editing can be mitigated by altering the pro
tein, whether in nCas9 or in the deaminase domain.
2.3. Prime editing
Prime editing, which is often likened to a word processor, offers the ad
vantages of base editing with respect to efficiency and precision while
overcoming the latter’s limitation of making only select single-nucleotide
changes.6,24
All possible nucleotide changes, as well as any small indel
mutations and even large deletions, lie within the scope of prime editing.
A version of nCas9 that nicks the non-target DNA strand (in contrast to
base editors, which nick the target DNA strand) is fused to a reverse
transcriptase (RT) (Figure 3). The gRNA has a 3′
extension that serves
two purposes—the distal end (primer-binding site, or PBS) hybridizes
with the non-target DNA strand upstream of the nick (i.e. starting
with the fourth nucleotide upstream of the PAM), and the intermediate
portion (reverse transcriptase template, or RTT) serves as a substrate
on which the RT can build a DNA flap directly attached to the non-
target DNA strand at the nick site. There is substantial flexibility with
respect to the length of the RTT and the mutations that can be incorpo
rated into the RTT—up to dozens of nucleotides and possibly even
longer—as well as positioning vis-à-vis a given PAM, offering an ex
panded targeting range compared to standard nuclease gene editing
and base editing. The prime editing gRNA (pegRNA) is vulnerable to
exonuclease action at its 3′
end, impairing the prime editing process,
and so the addition on the 3′
end of a structured RNA motif that is re
sistant to cleavage (engineered pegRNA, or epegRNA) can substantially
increase the efficiency of prime editing.25
Through a complex repair process, the mutation-bearing DNA flap
can displace and cause the excision of the local non-target DNA strand
downstream of the nick, followed by ligation that results in an intact
double-strand DNA in which the non-target strand has the desired mu
tation and the target strand has the original sequence. The mismatched
strands can be resolved in favour of either strand, but there are at least
two means by which to promote resolution in favour of the mutant
strand (resulting in the completion of prime editing). First, the same
nCas9-RT fusion protein can be used with a second, standard-length
gRNA with a spacer sequence that fosters a nick on the original strand;
nick repair then replaces the original nucleotides with nucleotides com
plementary to the mutant strand. Second, the cellular mismatch repair
(MMR) machinery tends to favour the original strand; accordingly, the
inhibition of MMR can increase the efficiency of prime editing, e.g. adding
a dominant-negative inhibitor of one of the MMR proteins to the
nCas9-RT protein.26
Similar to the use of two nucleases that introduce DSBs at sites flank
ing a region intended for deletion, prime editing with two pegRNAs can
engineer precise, large deletions.27,28
In what has been termed twin
prime editing, if the two pegRNAs generate flaps that contain comple
mentary sequences, they can be used to replace a DNA region with an
other DNA sequence encoded by the flaps.29
Although off-target mutagenesis with prime editing has been docu
mented, the full extent of off-target prime editing—in particular,
whether the use of RT poses any unexpected risks—remains to be clari
fied. If its safety proves to be favourable relative to other types of editing,
prime editing has the potential to become the preferred editing modality
for most applications in light of its versatility.
2.4 Epigenome editing
Epigenome editing is distinct from the other types of editing because it
does not involve any changes to DNA sequences. Rather, it modifies
gene expression by affecting how proteins interact with DNA se
quences. If a dCas9/gRNA complex is directed to a sequence in a
gene promoter or transcriptional enhancer (Figure 4), it will not directly
affect the DNA molecule but nonetheless can sterically interfere with
factors that normally interact with the sequence and thereby gene ex
pression (CRISPR interference).30
More potent knockdown can be
achieved if dCas9 is fused to a domain that actively suppresses gene ex
pression, such as the Krüppel-associated box (KRAB) domain, by modi
fying the local chromatin structure and thereby the accessibility of the
DNA sequence to the transcriptional machinery.31
Conversely, in
creased gene expression (CRISPR activation) can be achieved by fusing
dCas9 to a domain that acts as a transcriptional activator, such as VP16,
or by tethering activators to the gRNA by engineering RNA aptamers on
its 3′
end.32
These gene expression changes appear to be transient, in
that they persist only as long as the dCas9 fusion protein remains pre
sent at the site. More durable epigenome editing can be achieved if
dCas9 is fused to a methyltransferase or demethylase domain.33
Increased methylation in or near the promoter of a targeted gene, par
ticularly at cytosine bases in CpG dinucleotide sequences, typically re
sults in reduced gene expression, whereas decreased methylation
typically results in increased gene expression. Such methylation changes
have been observed to endure in cells through many divisions, and yet
the changes can be reversed by subsequently directing a dCas9 fusion
protein with the opposite effect to the same target genomic site.
3. Cardiovascular research
applications
3.1 Animal models
Perhaps the biggest impact of CRISPR on cardiovascular research—and
biomedical research generally—has been in facilitating the rapid gener
ation of genetically modified animal models. Standard nuclease gene edit
ing is well suited to the creation of knockout and knock-in mouse
models of disease in as little as three weeks (the length of mouse gesta
tion),34,35
so much so that it is supplanting the traditional means of gen
erating such models: in vitro modification of mouse embryonic stem cells
by homologous recombination, implantation into blastocysts to gener
ate chimeric mice, and a minimum of one round of breeding. NHEJ is
so efficient when SpCas9 is injected into zygotes (single-cell mouse em
bryos) that multiple genes can be fully knocked out simultaneously, sim
ply by co-injecting multiple gRNAs.34
The CRISPR components can be
delivered as DNA vectors, RNAs, or pre-assembled ribonucleoprotein
complexes. The introduction of indel mutations early in the coding se
quences of genes can result in relatively clean gene knockout, via the
dual mechanisms of severe truncation of protein products and
nonsense-mediated decay of messenger RNAs. NHEJ also permits dis
ruption of regulatory elements and, when using two gRNAs, precise de
letions in the genomes of zygotes.
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Not unexpectedly, HDR to precisely insert a mutation, tag, or report
er upon co-injection of SpCas9, gRNA, and a synthetic DNA repair tem
plate into mouse zygotes is typically much less efficient than NHEJ
mutagenesis. Nonetheless, just one correctly edited allele in the zygote,
yielding a heterozygous mouse, permits the breeding of a large colony of
mice with the desired genotype. Moreover, if the other allele in the zyg
ote undergoes undesired NHEJ editing, that allele can be bred out of the
colony; the same is true of any off-target edit unless it is close enough on
the same chromosome to co-segregate with the desired edit. It is pos
sible to simultaneously introduce multiple alterations (e.g. two loxP sites
flanking a portion of a gene) by co-injecting multiple gRNAs and multiple
repair templates, though success is infrequent.36
Alternatively, multiple
mutations can be introduced serially: one round of HDR to insert the
first mutation, followed by breeding of enough correctly targeted
mice to obtain zygotes for a second round of HDR to insert the second
mutation, etc.37
Along the same lines, it is quite feasible to start with zy
gotes from mice of any genetic background and use CRISPR to add add
itional mutations to that background.
A decisive advantage of zygote editing is that it can be applied widely
across species, in principle allowing for any kind of animal to serve as a
disease model. This versatility is especially pertinent to cardiovascular
diseases, which are often poorly modelled in mice. Models that better
phenocopy human diseases include rats, rabbits, pigs, and non-human
primates, all of which have proven to be quite amenable to CRISPR
modification. In one example, CRISPR nuclease editing to knock out
the DMD gene generated a pig model of Duchenne muscular dystrophy
that displayed skeletal and cardiac muscle defects.38
In another example,
CRISPR nuclease editing to knock out the SAP130 gene, which encodes a
Figure 4 Epigenome editing. The use of catalytically dCas9, with tethering of a regulatory domain or enzyme to either Cas9 or the guide RNA (or both),
can introduce chromatin changes or DNA methylation changes that result in transcriptional activation or interference with a target gene’s expression with
out alteration of the DNA sequence.
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chromatin modifier previously linked to congenital heart disease, yielded
pigs with tricuspid valve dysplasia or atresia.39
An alternative approach to zygote editing for the purpose of disease
modelling is somatic editing, in which the editing is undertaken in living
animals. Viral vectors, particularly adeno-associated virus (AAV) vectors,
have proven to be effective in delivering CRISPR–Cas systems into or
gans such as the liver and the heart. A disadvantage of AAV vectors is
the limited cargo size, roughly 4.7 kb, which cannot easily accommodate
the gene encoding SpCas9 (≈4.2 kb, not including the requisite pro
moter and polyadenylation sequence) and a gRNA expression cassette
(≈0.5 kb). One workaround is the use of a smaller Cas9 isoform such as
SaCas9 (≈3.3 kb) so that the entire CRISPR–Cas system can fit into a
single AAV vector.8
Another strategy is to split up the components
among multiple AAV vectors. SpCas9, as well as the larger fusion pro
teins used for base editing or prime editing, can be split into two parts
that upon delivery into cells with dual AAV vectors can spontaneously
assemble into a working protein via intein-mediated splicing (split-intein
strategy).40
Yet another strategy is to use an animal model that already
has the gene encoding the Cas protein within its cells—e.g. Cas9 trans
genic mice—allowing for straightforward delivery of a gRNA or multiple
gRNAs into the target cells with a single AAV vector.41
Finally, non-viral
approaches such as lipid nanoparticles (LNPs)42
and virus-like particles43
have the potential to deliver CRISPR–Cas systems as RNAs and ribonu
cleoprotein complexes, respectively, with less constraint on size, though
their organ-targeting range remains more limited than that of AAV
vectors.
In one example of somatic editing for disease modelling, CRIPSR nu
clease editing in a cardiac-specific Cas9 transgenic mouse sufficiently
knocked out the Myh6 gene in the heart to cause hypertrophic cardio
myopathy.44
In another example, CRISPR nuclease editing in a condi
tional Cas9 transgenic mouse model individually knocked out each of
nine different genes in the heart and demonstrated functions for
junctophilin-2 and ryanodine receptor-2 in T-tubule stabilization and
maturation, respectively.45
3.2 Human pluripotent stem cell models
Human pluripotent stem cells, particularly induced pluripotent stem
cells (iPSCs), are increasingly being used as substitutes or complements
to animal models of cardiovascular diseases, as they provide a ready
source of human cells for the study of disease phenotypes.46
iPSCs
have several advantages over immortalized or transformed cultured
cell lines: they have normal human karyotypes, they are genetically
matched to the person of origin (who is often selected due to their dis
ease status), and they can be differentiated into a variety of cell types
relevant to cardiovascular diseases, including cardiomyocytes, vascular
endothelial cells, smooth muscle cells, hepatocytes, and macrophages.
When one is studying the impact of a genetic variant on disease, an
important consideration for any experiments involving patient-derived
iPSCs is the choice of control iPSCs used to determine whether the for
mer have meaningful phenotypes. Simply using iPSCs from healthy indi
viduals as comparators render any apparent phenotypes susceptible to a
variety of confounders—differences in genetic background, sex, ethni
city, epigenetics, pluripotency, capacity to differentiate into the desired
cell type, method of derivation, and other characteristics. CRISPR editing
offers a means to reduce these confounders and isolate the effects of the
variant in question, by removing the variant while leaving the iPSCs
otherwise unchanged (isogenic cells). Alternatively, editing can introduce
a variant into wild-type iPSCs, which can be useful when iPSCs are not
available from a patient with that variant. Furthermore, editing can
generate an allelic series of variant cell lines that are all on the same gen
etic background, allowing for a rigorous comparison of the relative ef
fects of the variants.
As with animal models, edited iPSC lines have been used to study a
broad range of cardiovascular traits and diseases including cardiomyop
athies, lipid metabolism, vascular disorders, valvular disease, and arrhyth
mia disorders.46
Besides assessing the effects of pathogenic variants on
disease phenotypes, iPSCs have been used to dissect molecular mechan
isms by relating gene function to phenotypes (e.g. gene knockout or
knockdown), coding variants to protein function, and non-coding var
iants to gene expression.
Beyond their use in modelling studies to dissect disease mechanisms,
iPSCs are increasingly being employed for translational purposes.
Isogenic iPSCs with and without variants of uncertain significance, iden
tified in patients known or suspected to have inherited cardiovascular
diseases, can clarify whether the variants are pathogenic or benign.47,48
In one example, for a 65-year-old woman with hypertrophic cardiomy
opathy who was found to have a variant of uncertain significance in
TNNT2, isogenic iPSCs with the variant were generated and phenotyped
in ,3 months, in between the patient’s first and second genetics clinic
visits. The work ascertained the variant to be a likely benign variant,
which was communicated to the patient and her provider and incorpo
rated into the management plan in real time (e.g. advising against cascade
genetic testing for the variant in family members).48
An important caveat
is that the prognostic capabilities of iPSCs for variant classification re
main unproven. A different translational application of iPSCs is to gener
ate somatic cell types for which obtaining primary cells is prohibitive, e.g.
cardiomyocytes, for use as a test platform in which to assess the efficacy
and safety of CRISPR–Cas systems that are ultimately intended to be de
ployed for therapeutic uses in patients.
The most commonly used strategy for introducing or removing var
iants in iPSCs is standard nuclease gene editing with HDR, typically via
transfection or electroporation of a CRISPR–Cas system along with a
single-strand DNA oligonucleotide to serve as a repair template, often
followed by fluorescence-activated cell sorting (FACS) or transient anti
biotic selection to enrich for cells receiving the editing components (par
ticularly if using DNA vectors to deliver the CRISPR–Cas system), and
then clonal outgrowth of single cells. This approach is notable for its ex
treme locus-to-locus variability in efficacy. In some cases, the proportion
of correctly edited clones may exceed 10%, but in many cases, the pro
portion is ,1% and often much ,1%, requiring the screening of hun
dreds of clones. This phenomenon can present a substantial challenge
given that rigorous iPSC-based studies will ideally use multiple clones
of each genotype, to mitigate the consequences of an off-target edit in
any given clone or of stochastic clone-to-clone heterogeneity. The use
of a DNA vector with an embedded antibiotic resistance cassette, close
to the variant, as the repair template allows for sustained antibiotic se
lection that substantially increases the reliability and efficiency of editing;
but this strategy requires a subsequent manipulation to remove the cas
sette from the genome, typically with a scarless approach such as the
piggyBac transposon system.49
3.3 Genome-wide or targeted functional
screens
A cardinal advantage of CRISPR–Cas systems is that they can target nu
merous genomic sites in parallel. With SpCas9-based systems—
whether standard nuclease gene editing, base editing, prime editing, or
epigenome editing—targeting the protein to a different site is simply a
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matter of altering the first 20 nucleotides of the gRNA. Accordingly, it is
straightforward to clone a pool of as many as hundreds of thousands of
short oligonucleotides into a common plasmid backbone in order to
generate a library of gRNA-expressing vectors. In principle, this permits
the generation of lentiviral or AAV pools for infection of cells in vitro or in
vivo to screen for the effects of individual or combinatorial gene perturb
ation on a phenotype of interest. An individual academic laboratory can
readily make a library with multiple gRNAs targeting each of the ≈20
000 genes in the human genome or, alternatively, make a small library
that screens a defined subset of genes, e.g. those that encode kinases
or those that encode transcription factors.
Genome-wide and other large-scale CRISPR screens using standard
nuclease gene editing, CRISPR interference, CRISPR activation, and
base editing have become well-established methodologies.50
Standard
nuclease gene editing and CRISPR interference are complementary ap
proaches; gene knockout results in null alleles that can unmask other
wise hidden phenotypes but can disrupt essential genes, whereas
partial gene knockdown can reveal subtler phenotypes. CRISPR activa
tion provides a complementary approach to cDNA library screening
and overcomes the limitation that large genes are difficult to overex
press as cDNAs. Base editing can lend itself to gene knockout but can
also model the effects of a spectrum of variants ranging from dominant-
negative activity to gain of function. Designing a screen is a nuanced de
cision that entails consideration of a variety of factors: the type of editor;
the size and comprehensiveness of the library to be used; the cell type to
be studied; whether the targeted cells already stably express the Cas
protein, or whether the Cas and gRNA components are both delivered
via viral vectors; whether to perform the screen in vitro (much more
commonly done) or in vivo; the phenotype to be screened (typically a
self-selecting phenotype, like cell proliferation or survival upon exposure
to a drug, or a phenotype that can be identified through a marker or anti
body, permitting FACS); and whether to focus on positive selection
(identifying gRNAs enriched in the cells selected for analysis) or negative
selection (identifying gRNAs depleted in the cells) or both. Finally, any
hits that emerge from the primary screen need to be subjected either
to secondary screening or individual experimentation—ideally with
gRNAs distinct from the ones used for the primary screen—for valid
ation as true-positive findings.
One set of examples of productive CRISPR screens involved the
identification of modifiers of LDL cholesterol metabolism, particularly
the action of the LDL receptor—which resides at the cell surface and
moves LDL particles out of the bloodstream and into the cell—and
the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein—
which acts as an antagonist to LDL receptor by binding to it at the
cell surface and causing it to be internalized and degraded in the cell.
In the first study, a genome-wide CRISPR nuclease screen in HEK
293T cells stably expressing PCSK9 fused to green fluorescent protein
sought to identify modifiers of PCSK9 secretion.51
The most enriched
gRNAs in FACS-isolated cells with the highest amount of internal fluor
escence (inhibited PCSK9 secretion) all targeted the SURF4 gene, an
endoplasmic reticulum cargo receptor. Further investigation revealed
that SURF4 directly interacts with PCSK9 in the early secretory path
way, promoting the efficient export and secretion of PCSK9. Three sub
sequent studies sought to discover novel modifiers of cellular LDL
uptake. One study used genome-wide CRISPR nuclease screening in
HuH-7 human hepatoma cells and targeted secondary screening in
HuH-7 and HepG2 human hepatoma cells to identify enriched
gRNAs in cells with very high or very low uptake of fluorescently con
jugated LDL particles; among the novel hits were genes encoding
components of the exocyst, an octameric protein complex involved
in vesicular trafficking.52
Another study used genome-wide CRISPR nu
clease screening in HepG2 cells to identify enriched gRNAs in cells with
very low uptake of fluorescently conjugated LDL particles; follow-up in
dividual experimentation confirmed three hits—SYNRG, C4orf33, and
TAGLN.53
Subsequent investigation found transgelin (the protein prod
uct of TAGLN), which is an actin-binding protein, to be involved in LDL
receptor endocytosis. The third study used genome-wide CRISPR inter
ference (dCas9-KRAB) screening in HepG2 cells to identify enriched
gRNAs in FACS-isolated cells with high or low surface LDL receptor ex
pression; follow-up individual experimentation confirmed many hits and
focused attention on the top-performing hit, CSDE1, which was found
to promote the degradation of the LDLR messenger RNA.54
The com
plementary findings of these various studies demonstrate the benefit of
using orthogonal CRISPR-based approaches to answer similar biological
questions.
In one example of a targeted in vivo functional screen, AAV vectors
with gRNAs against a set of 2444 genes focused on transcription factors
and epigenetic modifiers were administered as a pool into neonatal
transgenic mice harbouring both SpCas9 and a Myh7-yellow fluorescent
protein reporter acting as a marker of cardiomyocyte maturation.55
After 1 month, FACS-isolated cardiomyocytes with high reporter ex
pression were assessed for either enrichment or depletion of gRNAs.
After individual experimentation with the top novel hits, the related
Rnf20 and Rnf40 genes emerged as positive regulators of cardiomyocyte
maturation. RNF20 and RNF40 are components of a ubiquitin ligase
complex that monoubiquitinates histone H2B on lysine 120, an epigen
etic mark that controls dynamic changes in gene expression required for
maturation.
4. Therapeutic applications
4.1 Hypercholesterolaemia
Therapeutic CRISPR editing entails two approaches: ex vivo editing, in
which a patient’s cells are extracted, edited outside of the body, and
transplanted back into the body; and in vivo somatic editing, in which
the editing tool is administered directly into the body. As a practical mat
ter, it is unlikely that for the cell types most relevant to cardiovascular
diseases—cardiomyocytes, hepatocytes, vascular cells, etc.—ex vivo
editing and re-transplantation will be a viable approach for the foresee
able future. As such, this section focuses on emerging in vivo somatic
editing applications that target the liver or the heart. Three diseases,
in particular, stand out as eminently addressable by CRISPR editing in hu
man patients, with clinical trials underway or on the horizon: hyperchol
esterolaemia, transthyretin amyloidosis, and Duchenne muscular
dystrophy.
The best validated genetic targets for the treatment of hypercholes
terolaemia are the gene encoding the aforementioned PCSK9 protein
and the angiopoietin-like 3 (ANGPTL3) gene.56
For either gene, single
loss-of-function variants result in reduced LDL cholesterol levels and
in protection against coronary heart disease without any serious adverse
health consequences, and double loss-of-function variants (complete
gene knockout) have been observed in healthy individuals. Both genes
are largely expressed in the hepatocytes, and the protein products are
secreted into the bloodstream. Monoclonal antibodies against either
protein result in substantially reduced LDL cholesterol levels—up to
≈60%—in hypercholesterolaemic patients and have been approved
for medical use, and short-interfering RNAs and antisense
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oligonucleotides are in clinical trials. A limitation common to these treat
ments as well as to the mainstay of lipid-lowering medications, the sta
tins, is that they must be taken repeatedly for the lifetime (whether
every day, every few weeks, or every few months) in order to achieve
the full therapeutic benefit. CRISPR editing of either PCSK9 or
ANGPTL3 in the hepatocytes would potentially confer the same thera
peutic benefit with a single course of treatment, i.e. a ‘one-and-done’ so
lution to hypercholesterolaemia, due to the permanence of changes to
the DNA sequence.
In an early study, an adenoviral vector encoding SpCas9 and a gRNA
targeting a sequence in exon 1 of the mouse Pcsk9 gene was adminis
tered to mice in order to knock out Pcsk9 alleles in hepatocytes via
NHEJ.57
Although adenoviral vectors are generally not used for genetic
therapies due to the risk of life-threatening immune responses, one was
used in this proof-of-concept study to accommodate the large size of
SpCas9. Several days after receiving the treatment, the mice had
.50% whole-liver editing at the Pcsk9 target site. Consistent with
NHEJ repair, the most common edits were 1 or 2 bp deletions or inser
tions, although deletions as large as dozens of base pairs were evident.
Consistent with the high degree of editing, there were reductions in
blood PCSK9 protein levels of ≈90% and of blood cholesterol levels
of ≈40%, nearly as large as cholesterol reductions observed in germline
Pcsk9 knockout mice. Screening of a handful of candidate off-target sites,
chosen by high sequence similarity to the Pcsk9 target site, showed no
evidence of editing. A subsequent study using an adenoviral vector
with SpCas9 and the same Pcsk9 gRNA confirmed the therapeutic effect
on cholesterol levels and performed a much more rigorous assessment
of off-target editing, using a two-stage approach.58
First, a biochemical
technique called CIRCLE-seq—in which circularized mouse genomic
DNA fragments were mixed with SpCas9 protein and the Pcsk9
gRNA in vitro, followed by next-generation sequencing to identify any
DNA fragments that were linearized by SpCas9 cleavage—identified
182 candidate off-target sites. Second, PCR amplification of each of
the candidate sites from liver genomic DNA samples obtained from
the treated mice, followed by deep next-generation sequencing, showed
no evidence of off-target editing in vivo.
While these studies established the efficacy and off-target safety of
Pcsk9 editing in mice, the findings have limited relevance to a potential
PCSK9-editing therapy in human patients. First, the DNA sequences of
the mouse Pcsk9 gene and the human PCSK9 gene have substantial dif
ferences, and so even if the same exon 1 site were to be targeted in hu
man hepatocytes, the gRNA spacer sequence would be distinct and
might not support the same efficacy of editing. Second, the favourable
off-target profiling observed in the mouse genome might not hold up
in the human genome, due to the profound differences between the
two genomes. An independent assessment would need to be performed
in human hepatocytes. Third, the significant physiological differences be
tween mouse and human hepatocytes could mean that even if the same
gRNA were being used to target the same target site, the editing out
comes could very well differ between the two species. To establish a
preclinical model system in which to better test a human-directed ther
apy, chimeric liver-humanized mice—a xenograft model in which the
mouse hepatocytes are replaced with transplanted primary human he
patocytes that engraft in the liver—received an adenoviral vector encod
ing SpCas9 and a gRNA targeting a sequence in exon 1 of the human
PCSK9 gene.59
There was ≈50% editing of the PCSK9 gene within the hu
man hepatocytes in the liver, predominantly small indel mutations, and a
reduction of human PCSK9 protein levels in the blood of ≈50%.
Screening of a handful of candidate off-target sites showed no evidence
of editing. A subsequent study performed a much more rigorous assess
ment of off-target editing in the liver-humanized mice treated with the
adenoviral vector.60
First, a biochemical technique called ONE-seq—
in which tens of thousands of synthetic DNA oligonucleotides matching
genomic sites selected for their high sequence similarity to the PCSK9
target site were mixed with SpCas9 protein and the PCSK9 gRNA in vitro,
followed by next-generation sequencing to identify any cleaved DNA
fragments—rank-ordered all of the tested sequences by cleavage activ
ity. Second, PCR amplification of each of the 40 top-ranked sites from
liver genomic DNA samples obtained from the treated mice, followed
by deep next-generation sequencing, showed off-target editing at four
of the sites in vivo, arguing against further development of the human-
specific gRNA used in these studies into a therapy.
A separate set of studies turned to delivery methods more amenable
for use in human therapeutics than adenoviral vectors. Taking advantage
of the small size of SaCas9 compared with SpCas9, one study adminis
tered to mice an AAV vector encoding SaCas9 along with either of two
gRNAs targeting the mouse Pcsk9 gene, with the intent of using NHEJ to
disrupt the gene in the liver.8
Similar to the aforementioned adenoviral
mouse studies, AAV treatment resulted in 40–50% whole-liver Pcsk9
editing, with small indel mutations being the most frequent editing out
come, and reductions of blood PCSK9 protein levels of .90% and of
blood cholesterol levels of ≈40%. There was no evidence of off-target
editing at a handful of candidate sites in liver genomic DNA samples
from treated mice. The success of AAV-mediated somatic nuclease
gene editing in mice was followed by an exploration of non-viral meth
ods to deliver CRISPR–Cas systems into the mouse liver. One study seri
ally injected LNP formulations with in vitro transcribed SpCas9
messenger RNA or a chemically synthesized gRNA targeting a sequence
in the mouse Pcsk9 gene.61
The LNP treatments resulted in reductions
of PCSK9 protein levels in the liver of 40%–50%. A more comprehensive
study co-injected LNP formulations with either the SpCas9 messenger
RNA or a mix of two gRNAs targeting distinct sequences in Pcsk9,
each of which had chemical modifications intended to enhance RNA sta
bility in vivo.42
The LNP treatment resulted in .80% whole-liver editing
of the gene, with NHEJ-mediated deletion between the two gRNA tar
get sites being the most frequent editing outcome. The editing was ac
companied by an absence of detectable blood PCSK9 protein and a
reduction of blood cholesterol levels of ≈40%. No Pcsk9 editing was evi
dent in the lungs or spleen, suggesting that the LNPs preferentially tar
geted the liver, the Pcsk9 locus was accessible to nuclease action only in
liver cells, or both. A subsequent study treated mice with a single LNP
formulation with both SpCas9 messenger RNA and a gRNA targeting
a sequence in the mouse Angptl3 gene and achieved ≈40% whole-liver
Angptl3 editing and a reduction of blood ANGPTL3 protein levels of
≈65%.62
An even more recent study used engineered virus-like particles
to deliver CRISPR ribonucleoproteins into the mouse liver, resulting in
≈63% whole-liver Pcsk9 editing and a reduction of blood protein
PCSK9 levels of ≈78%.43
Yet another series of studies explored the use of CRISPR technolo
gies besides standard nuclease gene editing. Cytosine base editors can
knock out genes either through the direct introduction of nonsense mu
tations via C-to-T changes on the sense strand (CAG→TAG,
CAA→TAA, CGA→TGA) or C-to-T changes on the antisense strand
(TGG→TAG, TGG→TGA, TGG→TAA); the disruption of the start co
don (ATG→ATA); or the disruption of a canonical splice donor
(GT→AT) or splice acceptor (AG→AA). In contrast, adenine base edi
tors are restricted to disruption of the start codon (ATG→GTG,
ATG→ACG), a splice donor (GT→GC), or a splice acceptor
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(AG→GG). One study administered to mice an adenoviral vector en
coding a cytosine base editor and a gRNA targeting the Pcsk9
tryptophan-159 codon (TGG), with the intent to introduce nonsense
mutations.63
The base-editing treatment resulted in ≈30% whole-liver
editing of the gene, with nonsense mutations being the predominant
editing outcome, although low levels of bystander missense mutations
and indel mutations were evident as well. There were reductions of
blood PCSK9 protein levels of ≈60% and of blood cholesterol levels
of ≈30%. A separate study used an adenoviral vector encoding a cyto
sine base editor and a gRNA targeting the Angptl3 glutamate-135 codon
(CAA), resulting in ≈35% whole-liver Angptl3 editing and reductions of
blood ANGPTL3 protein levels of ≈50% and of blood cholesterol levels
of ≈20% in wild-type mice.64
The therapeutic effects were accentuated
in LDL receptor knockout mice (which phenocopy the severest form of
inherited hypercholesterolaemia, homozygous familial hypercholester
olaemia), with reductions of blood cholesterol levels of ≈50% and of
blood triglyceride levels of .50%. A study of epigenome editing
co-administered to mice two AAV vectors encoding catalytically dead
SaCas9-KRAB and a gRNA targeting a sequence in the mouse Pcsk9 pro
moter, respectively, resulting in reductions of whole-liver Pcsk9 gene ex
pression of ≈50% and of blood PCSK9 protein levels of 80%, with
concordant reduction of blood cholesterol levels.65
The therapeutic ef
fects appeared to attenuate over the course of 6 months, suggesting that
expression of the Cas protein was waning over time. A study of prime
editing used two AAV vectors encoding a split-intein prime editor to
achieve the insertion of a TGA stop codon into Pcsk9 exon 1, albeit at
low efficiency, ≈13% whole-liver editing.66
Recently, two studies addressed the efficacy and safety of in vivo somatic
editing in non-human primates, specifically cynomolgus monkeys.67,68
Both
studies used adenine base editors in combination with the same gRNA tar
getingthesplicedonorattheendofPCSK9exon1,withthegoalofknocking
outthegenebyincorporatingsomeorallofintron1inthemessengerRNA.
ThegRNAmatchesboththemonkeyandhumanPCSK9sequences;fortuit
ously, readthrough into human PCSK9 intron 1 leads to a ribosome quickly
encountering a stop codon, resulting in the production of a truncated pro
tein spanning the codingsequence of exon1and justthree additionalamino
acids.Ofnote,thegRNA-targetedhumanPCSK9sequencehasminimalnat
urallyoccurringgeneticvariation(conservedin.99.9%ofcataloguedalleles
across populations), which is advantageous for the development of an edit
ingtherapeutic.EachstudyadministeredasingleLNPformulationwithboth
an adenine base editor messenger RNA and the gRNA into monkeys via
intravenous infusion. In one study, the therapeutic effects were relatively
modest, with 26% whole-liver PCSK9 editing and reductions of blood
PCSK9 levelsof 32% and of blood LDL cholesterol levelsof 14% at1 month
aftertreatment.67
Intheother study,thetherapeutic effectsweremuch lar
ger and more compatible with clinical translation, with 66% whole-liver
PCSK9 editing and reductions of blood PCSK9 levels of ≈90% and of blood
LDL cholesterol levels of ≈60% persisting for more than 8 months in an on
going study.68
The predominant editing outcome was the desired A-to-G
single-nucleotide change, with low levels of indel mutations.
With respect to safety, the monkeys in the two studies tolerated the
LNP treatment well, without any adverse clinical events. The lipid and
mRNA components of the treatment were found to be cleared within
2 weeks. Although there were immediate, transient rises in blood trans
aminase levels after treatment, these changes resolved in 1–2 weeks, and
there was no subsequent transaminitis to suggest a cytotoxic immune
response. In the first study, some animals received multiple infusions
of LNPs and developed antibodies against the adenine base editor,
though whether this phenomenon would affect the efficacy of future
LNP treatments is unclear. Both studies assessed for off-target editing,
with the first study using CIRCLE-seq and the second study using
ONE-seq (among other methods) to identify candidate sites.
Although there was low-level off-target editing at one candidate site in
monkey liver (at a site not conserved in the human genome), there
was no evidence of off-target editing in cultured human hepatocytes, in
cluding gRNA-independent editing by the deaminase domain of the base
editor. In the light of these preclinical studies, PCSK9 somatic editing ap
pears to be poised to enter clinical trials in the near future.
4.2 Transthyretin amyloidosis
As recounted in the opening clinical vignette, TTR somatic editing for the
treatment of transthyretin amyloidosis has already met early success in a
clinical trial. Similar to PCSK9 and ANGPTL3, the TTR protein is largely
expressed in the hepatocytes and secreted into the bloodstream. TTR
normally forms tetramers that function as thyroxine and vitamin A trans
porters; destabilized TTR monomers have the potential to form abnormal
aggregates that accumulate and damage the heart (cardiomyopathy) or
nerves (polyneuropathy) or both, causing disease with a high degree of
morbidity and mortality. The aggregates are more likely to form from mu
tant protein, driving the disease process at an earlier age, but can also form
from wild-type protein, causing disease late in life. Accordingly, knockdown
of TTR expression in hepatocytes can address disease caused by either mu
tant or wild-type protein. A short-interfering RNA and an antisense oligo
nucleotide, each targeting the TTR messenger RNA and reducing blood
TTR protein levels by ≈80%, have been demonstrated in trials to confer
clinical benefit and have been approved for medical use.69,70
Preclinical studies in mice and non-human primates administered
LNPs with SpCas9 messenger RNA and a gRNA targeting the Ttr or
TTR gene, in each species achieving up to ≈70% whole-liver editing—
producing the expected indel mutations via NHEJ—and .95% reduc
tions of blood TTR protein levels that persisted for a year in long-term
studies.71,72
In an ongoing clinical trial, one group of three patients re
ceived a very low dose (0.1 mg/kg) of LNPs with SpCas9 messenger
RNA and a gRNA targeting exon 2 of the human TTR gene, and a second
group of three patients received a low dose (0.3 mg/kg) of the LNPs.72
One month after treatment, the first and second groups of patients ex
perienced mean 52 and 87% reductions of TTR levels, respectively, with
one of the patients in the second group having a remarkable 96% reduc
tion—outstripping the therapeutic effects of the short-interfering RNA
and antisense oligonucleotide therapies. None of the patients had trans
aminitis or any serious adverse events from the treatment. The durabil
ity of the patients’ TTR reductions over time, as well as the effects of
higher LNP doses that are being tested in additional patients in the trial,
remain to be seen. Nonetheless, this trial stands out as the first success
ful demonstration of somatic in vivo editing of any kind in human patients.
4.3 Duchenne muscular dystrophy
Besides transthyretin amyloidosis and hypercholesterolaemia, the car
diovascular disease for which a somatic editing therapy appears to be
closest to the clinic is Duchenne muscular dystrophy (DMD); although
its hallmark is dysfunction of the skeletal muscles, a primary cause of
death is cardiac failure. Severe DMD arises from any a plethora of
exon deletion, frameshift, nonsense, or splice-site mutations in the
DMD gene on the X chromosome, with the majority of the mutations
residing in a hotspot spanning from exons 45 to 53 and disrupting the
protein product, dystrophin. Dystrophin is a structural protein that is
part of a complex that strengthens myofibres by linking cytoskeletal pro
teins to the cell membrane and extracellular matrix. Notably, dystrophin
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retains much of its function even if several of its middle exons are miss
ing, as long as the entire coding sequence is in the frame. This property
allows for therapeutic approaches in which one or more exons are
skipped in order to compensate for any of the aforementioned DMD
mutations, rather than direct correction of the pathogenic mutation.
One such approach is to use standard nuclease gene editing with two
gRNAs to effect NHEJ-mediated deletion of an exon or exons, and an
other approach is to use standard nuclease gene editing to disrupt a
splice site or splicing regulatory site to force the exclusion of an exon
from the Dmd transcript. These approaches are mutation-agnostic
and thus can be applied to entire subsets of DMD patients.
Unlike the liver, the only effective and clinically relevant approach
available for systemic delivery of an editing tool into the skeletal muscles
and heart in vivo is the use of AAV vectors. Four early proof-of-concept
preclinical studies of CRISPR editing to treat DMD used the same mouse
model, the mdx mouse, which has a nonsense mutation in exon 23 of the
Dmd gene. Two of the studies employed intravenous, systemic adminis
tration of dual AAVs in which one vector expressed SaCas9 and the
other vector expressed two gRNAs targeting sites flanking exon
23.73,74
Another study systemically administered a single AAV vector
with SaCas9 and two gRNAs to engineer deletion of exons 21, 22,
and 23.75
The other study used dual AAVs in which one vector ex
pressed SpCas9 and the other vector expressed a gRNA that targeted
the mutant sequence in exon 23 and a gRNA targeted towards the 3′
end of the exon, preventing the exon’s inclusion in the Dmd transcript.76
All four of these studies demonstrated low levels of editing that none
theless led to substantial enough restoration of dystrophin expression
in the skeletal muscles and heart to achieve significant improvements
in muscle function.
In a long-term mouse study that assessed mdx mice following systemic
treatment with dual AAVs expressing SaCas9 and two gRNAs targeting
sites flanking exon 23, Dmd editing and dystrophin expression persisted
in the skeletal muscles and heart for a year.77
Notwithstanding these en
couraging results, treated adult mice but not neonatal mice developed
antibodies as well as T-cell responses against SaCas9. Notably, an un
biased assessment of editing outcomes at the Dmd target site found
that integration of AAV vector sequences was the most frequent
event—an observation mirroring those of previous studies in which
AAV sequences were observed to efficiently incorporate into the sites
of DSBs—raising the possibility of unexpected genotoxic effects, though
no adverse consequences were noted during the course of this study.
The potential liabilities of foreign DNA integration inherent in the use
of AAV vectors in combination with standard nuclease gene editing
can be mitigated through the use of CRISPR tools that do not induce
DSBs. Adenine base editing has proven to be an effective alternative
to standard nuclease gene editing for the treatment of DMD in mouse
models. In a mouse model with a deletion of exon 51 of the Dmd
gene, a split-intein strategy was used to deliver an adenine base editor
via two AAV vectors, targeting and disrupting the splice donor site of
exon 50.78
The editing resulted in the skipping of exon 50, bringing
the in-frame combination of exon 49 and exon 52 together and rescuing
protein function. When administered in vivo via intramuscular injection,
the treatment partially restored dystrophin expression in myofibres. In
another study, an adenine base editor delivered intramuscularly using a
dual-AAV trans-splicing approach was able to directly correct a non
sense mutation in exon 20 of the Dmd gene at a low frequency and mod
estly restore dystrophin expression—a notable result, since it points to
the possibility of treating genetic diseases in which single-nucleotide mu
tations cannot be mitigated through exon-skipping approaches.79
Three studies have progressed beyond mouse models to demon
strate proof of concept of somatic editing treatments for DMD in large
animal models. In a dog model of DMD harbouring a splice site mutation
that results in the exclusion of exon 50, intravenous administration of an
AAV vector expressing SpCas9 and an AAV vector expressing a single
gRNA targeting a site near the 5′
end of exon 51 induced both small
frameshift mutations—many of which restored the reading frame of
the transcript—and deletions affecting an exonic splicing enhancer—
which resulted in skipping of exon 51 in addition to exon 50 and thereby
restored the reading frame.80
The editing resulted in partial restoration
of dystrophin expression in the heart and skeletal muscles throughout
the body. In a pig model of DMD lacking exon 52, administration of
dual AAV vectors expressing split-intein SpCas9 and two gRNAs target
ing sites flanking exon 51, thereby deleting exon 51 and restoring the
reading frame, partially restored dystrophin expression in the muscles
and improved skeletal muscle function and mobility.81
The treatment
also increased the lifespan of the pigs by reducing cardiac arrhythmo
genic vulnerability and staving off the sudden cardiac death that was
the primary cause of mortality in this DMD model. Balancing the posi
tive results of these studies is a more recent study in DMD dog models
that demonstrated that the therapeutic effect of AAV-SpCas9 in re
storing dystrophin expression was accompanied by substantial
SpCas9-specific humoral and cytotoxic T-lymphocyte responses that
resulted in muscle inflammation and in attenuation of dystrophin ex
pression over time, despite the use of immunosuppression.82
The use of AAV vectors to systemically deliver editing tools to the
skeletal and cardiac muscles in vivo in human patients will entail overcom
ing substantial challenges, such as pre-existing immunity preventing
AAV treatment,83
the induction of immune responses that limit the ef
fectiveness of treatment and prevent re-treatment,82
and the current
need for large AAV doses that can induce life-threatening liver toxici
ties. Further development of AAV technologies—such as the directed
evolution of novel muscle-tropic AAV serotypes that avoid the liver
and allow for substantially lower dosing84
—and methods to mitigate
any treatment-associated immune responses will be needed to bring
safe and effective DMD editing therapies within reach.
4.4 Additional considerations for
therapeutic applications
Although therapeutic applications of CRISPR technologies appear to
have much promise, commentators frequently raise two concerns.
First, potential genotoxicity is a major barrier to the deployment of
CRISPR editing therapies. This remains a purely theoretical concern,
as there have been no reports of unintended adverse clinical conse
quences specifically from off-target editing in any treated animal models
or in any of the patients who have received somatic editing therapies in
clinical trials to date. As such, regulatory agencies must balance the the
oretical risks of editing against the projected benefits of editing therapies
—with relatively more to be gained for grievous genetic disorders with
substantial unmet need, and relatively less to be gained for milder dis
eases for which there are already approved therapies—in deciding
whether to allow clinical trials to commence. Second, there could be po
tential downsides to ‘one-and-done’ therapies: what is beneficial at the
time of treatment might become detrimental at a later time in life (e.g. an
antihypertensive therapy that permanently reduces blood pressure), and
there might be moral hazard incurred by treatment (e.g. a therapy to
permanently reduce LDL cholesterol levels and future risk of coronary
heart disease could encourage some patients to thereafter make
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unhealthy lifestyle choices). This concern relies on the assumption that it
will be impossible to undo the effects of an editing therapy, when in fact
several editing modalities—base editing, prime editing, and epigenome
editing—lend themselves to direct, precise reversal in the future should
the need arise.
5. Conclusion
CRISPR editing unequivocally has transformed biomedical research and
promises to do the same for cardiovascular medicine, ushering in a new
paradigm of ‘one-and-done’ therapies to tackle common conditions like
coronary heart disease and rare genetic disorders alike. The prolifer
ation of CRISPR technologies in the decade since the first demonstra
tions of CRISPR-based mammalian gene editing in 2012 promises even
more explosive growth to come in the next decade—to the ultimate
benefit of scientists and patients everywhere.
Conflict of interest: K.M. is an advisor to and holds equity in Verve
Therapeutics and Variant Bio.
Data availability
As this is a review article, there are no data to make available.
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