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