Targets for Gene Therapy
Effective gene therapy aims to identify and counteract AF mechanisms
originating from or kindled by a genetic element.73Two principal driving mechanisms of AF are focal ectopic firing and
re-entry. Both of these mechanisms are dependent on electrical and
structural remodeling, autonomic nerve remodeling and
Ca2+-handling abnormalities.74Electrical remodeling is typically characterized by shortening of the
atrial action potential duration (APD) through a decrease in the L-type
Ca2+ current and an increase in the inward-rectifier
current (I K1), and the emergence of
constitutively active acetylcholine induced potassium current
(I KACh).2 Structural remodeling
results in left atrial enlargement, atrial fibrosis, and gap junction
remodeling, culminating as slow and heterogeneous
conduction.75 In this review, we will limit ourselves
to the current state of mechanistic targets utilizing aforementioned
gene therapy vectors.
Ion channels
Ion channels have long been a pharmacologic target for rhythm
management, so it follows that gene therapy would pursue a similar path.
Indeed, transfection of plasmid containing a clarithromycin-responsive
variant of KCNE2 (Q9E), encoding the IKr regulatory subunit, hMiRP, lead
to prolongation of the APD by administration of clarithromycin 2 weeks
later.76 Epicardial gene painting of adenovirus
containing a dominant-negative variant of KCNH2-G628S (encoding alpha
subunit of IKr) resulted in APD prolongation and reduction of AF burden
and inducibility in a porcine model of AF.77Similarly, Soucek et al. confirmed prolongation of APD with myocardial
injection and electroporation of adenoviruses expressing same KCNH2
variant in a canine model of AF.78 Genetic suppression
of TASK-1 (Tandem of P Domains in a Weak Inward Rectifying K+
Channel–Related Acid-Sensitive K+ Channel-1; K2P3.1) through
transfection of AAV containing atrial anti–TASK-1 siRNA lead to
reduction of expression of TASK-1 and prolongation of atrial APD and
refractoriness.79
Ca2+ handling
proteins
Abnormal sarcoplasmic reticulum (SR) Ca2+ leak via the
ryanodine receptor type 2 (RyR2) has been described in atrial
cardiomyocytes from AF patients and in various AF
models.80,81 This disruption in calcium handling
contributes to ectopic atrial activity and is implicated in the
progression from paroxysmal to persistent AF. Phosphorylation at the
residue site S2814 was shown to promote AF in mouse models, with mice
harboring a phospho-resistant RyR2 form (S2814A) exhibiting a reduced
susceptibility to AF.82,83 This was demonstrated
across two different mouse models of atrial arrhythmias: 1) mice lacking
the RyR2-stabilizing subunit FKBP12.6, which causes spontaneous
Ca2+ waves and leads to a higher incidence of
spontaneous and pacing-induced AF; and 2) mice exhibiting cardiac
overexpression of the transcriptional repressor CREM-IbΔC-X (CREM-TG),
which leads to atrial myopathy and spontaneous AF that progresses from
paroxysmal to persistent. Given these findings, gene therapy integrating
a phospho-resistant form of RyR2, such as RyR2-S2814A may be indicated
as a clinical target of interest.
Calmodulin (CaM) is an important regulator of RyR2. When bound to
Ca2+, CaM contributes to inactivation of RyR2. This
regulatory property was investigated in a mouse model of
catecholaminergic polymorphic ventricular tachycardia (CPVT), a syndrome
where shortened refractoriness of RyR2 plays a dominant
role.84,85 Liu et al. engineered a form of CaM with
slowed Ca2+ dissociation (CaM M37Q, or therapeutic
T-CaM).86 They showed that injection of AAV9 T-CaM
attenuated diastolic Ca2+ waves and prevented
ventricular tachycardias in a calsequestrin-associated mouse model of
CPVT. It is conceivable that gene therapy with T-CaM in the atria would
attenuate the SR Ca2+ leak, and may therefore reduce
atrial triggers and progression from paroxysmal to persistent AF.
Autonomic nerve
remodeling
The atria are highly innervated by the autonomic nervous system. Vagal
stimulation results in shortening of the atrial effective refractory
period (ERP) and increased vulnerability to AF.87Acetylcholine (ACh) released from parasympathetic nerves activates
muscarinic type 2 receptors which interact with heterotrimeric G
proteins: the Gαi/o subunits subsequently inhibit
adenylate cyclase protein kinase, and the Gβγ subunit activates
IKACh.88 Despite the apparent
importance of the autonomic nervous system in AF, drug therapy studies
using β-blockers and selective IKACh blockers have shown
modest success.89,90 Donahue et al. pioneered gene
therapy targeting specific components of the G-protein autonomic pathway
in the pig AV node as a rate control strategy for ventricular response
in AF. In the study, an adenoviral vector encoding for the G-protein
alpha inhibitory subunit 2 (Gαi2) was delivered in the
AV node of pigs, thereby mimicking increased vagal tone. There was a
substantial increase in the local expression of Gαi2 and
a slowing of conduction through the AV node.91Similarly, Murata et al. overexpressed the ras-related small G-protein
GEM in ovine AV node and showed slower conduction through AV node and
reduction of overall heart rate during AF. 92Conversely, another approach to AF rate control is the knockdown of the
stimulatory G protein α subunit (Gαs), which mimics
beta-blockade. Lugenvil et al. found that genetic inhibition of
Gαs protein using adenovirus containing siRNA against
Gαs in the AV node reduced heart rate by 20% and
prevented AF-associated cardiac dysfunction in a porcine
model.93 Our group also targeted of vagal signaling in
the left atrium by inhibiting Gαi and
Gαo in canine models.94 Here,
injection of plasmids encoding the inhibitory peptides of
Gαi and Gαo to multiple sites in the
posterior left atrium (PLA) lead to attenuation of vagal-induced
shortening of ERP and diminished AF inducibility during vagal
stimulation.94
Gap Junction remodeling
Connexins (Cxs) are subunit transmembrane proteins that oligomerize to
construct a connexon, composed of six Cxs. Gap junctions are formed as
the connexons of two neighboring cells dock together, permitting direct
cell-cell communication and bidirectional passage of ions and small
molecules up to 1 kd.95 Reduced expression or abnormal
localization of Cx40 and Cx43 are associated with impaired electrical
conduction in the atrium and an increased risk of developing
AF.96,97 Accordingly, gene transfer of both of these
connexins using an epicardial painting approach significantly improved
expression and localization of the proteins, and was associated with
improved conduction and a reduction in arrhythmia burden in a porcine
model of AF.98 A separate study of Cx43 alone in the
same type of model resulted in similar findings, with a marked reduction
in the development of persistent AF.99
Structural remodeling
Atrial fibrosis is also a well-known factor in the pathogenesis of AF,
and may explain the increasing prevalence of this arrhythmia with age. A
central feature of age-related fibrosis is up-regulation of transforming
growth factor (TGF)-β.100 The PLA has been found to
play an important role in the maintenance of AF due to increase in
susceptibility to fibrosis and inhomogeneous
conduction.101
Our group evaluated the effect of a transgene that interferes with TGF-β
signaling on structural remodeling in the PLA. Injection of a minigene
expressing a dominant-negative type II TGF-β receptor in the PLA of a
canine HF model of AF resulted in decreased fibrosis and reduction in
pacing-induced AF in the treated animals.13
Inflammation/Oxidative
injury
AF is a multifactorial disease and there is an ample evidence supporting
the involvement of inflammation and oxidative injury in the
pathophysiology of AF.102,103 Inflammatory processes
have been shown to affect the electrical and structural properties of
the atria.104 The importance of the NLRP3 (NACHT, LRR
and PYD domains-containing protein 3) inflammasome in the development of
AF was recently established. The activity of NLRP3 inflammasomes is
altered not only in patients with AF but also in canine RAP model and in
a murine model of spontaneous AF (CREM-TG mice), suggesting a major role
for NLRP3 inflammasome in AF pathophysiology in the context of different
pathologies. Yao et al. found that pharmacological inhibition by MCC950,
an AAV9-mediated shRNA delivery to knockdown NLRP3, or genetic
inhibition by NLRP3 knockout prevented the development of
AF.105
Oxidative injury results from the imbalance between the generation and
neutralization of reactive oxygen species (ROS), is a major contributor
for AF and a possible therapeutic target.106,107 ROS
generated in the cardiovascular system are primarily derived from NADPH
oxidase (NOX), mitochondrial electron transport chain, xanthine oxidase
and uncoupled nitric oxide (NO) synthase.108,109Despite considerable evidence that ROS play an important role in the
generation of AF, clinical trials using conventional antioxidants for
post-operative AF have been unsuccessful,110,111likely because antioxidants do not reach sufficient, localized
concentrations to overcome kinetic limitations and allow for scavenging
of highly reactive free radical species.112 Our group
recently demonstrated a clear causative role of NOX2-generated oxidative
injury in the genesis as well as the maintenance of AF. We showed that
oxidative injury contributes to electrical remodeling in AF by
upregulating a constitutively active form of acetylcholine-dependent
K+ current (I KAch) – calledI KH - by a mechanism involving frequency
dependent activation of protein kinase C epsilon (PKCε).
Injection and electroporation of plasmids expressing shRNA against NOX2
in the atrium of a canine AF model not only delayed the time to onset of
non-sustained AF more than 5 fold but also prevented the development of
sustained AF for up to 12 weeks.113
Apoptosis
Apoptosis is associated with inflammatory pathways which contribute to
electrical and structural remodeling in AF.104Downregulation of caspase-3 in canine AF model indicated association of
apoptosis with AF via inhibition of calpain, a intracellular
Ca2+ activated protease.114 Genetic
knockdown of caspase-3 by transfer of adenovirus containing siRNA
against caspase-3 suppressed or delayed the onset of persistent AF by
reduction in apoptosis and prevention of conduction delay in porcine
model. 115
MicroRNAs are a class of endogeneous non-coding small RNAs that are
becoming more recognized to play an important role in pathogenesis of
AF. Zhang et al. examined differential expression of miRNA in ganglionic
plexus of a canine AF model and found expression of miR-206 was elevated
10 fold and lentiviral infection of miR-206 resulted in repression of
superoxide dismutase-1 (SOD-1).116 Anti-miR-206
infection with lentiviral vector, thus, lead to prolongation of ERP and
reduction of AF inducibility.116