Optimization and Evaluation of Engineered Prokaryotic Sodium Channel Gene Therapy for Heart Failure and Cardiac Arrhythmias

Abstract

Despite continued progress, therapies to augment contractile function and prevent arrhythmias in patients with ischemic and non-ischemic heart disease remain limited. With growing understanding of the complex molecular mechanisms underlying cardiac function and dysfunction, gene therapies have emerged as a promising strategy to treat and potentially cure heart diseases. Particularly, therapies for cardiac arrhythmias and heart failure could greatly benefit from approaches that can augment peak cardiac Na+ or Ca2+ current in cardiomyocytes (CMs) by stably overexpressing mammalian voltage-gated Na+ or Ca2+ channels. However, these channels are encoded by large (>6kb) genes that cannot be packaged in adeno-associated viral vectors (AAV; limit 4.7kb), currently a standard delivery vehicle for stable expression of exogenous genes in the heart. Unlike their mammalian counterparts, prokaryotic voltage-gated sodium channels (BacNav) are homotetrameric proteins encoded by genes that are <1kb in size, suitable for packaging in any type of recombinant viral vector, including AAV. Thus, the objective of this thesis has been to optimize and evaluate BacNav gene therapy as a novel strategy for the treatment of heart failure and cardiac arrhythmias. Towards this goal, we first performed proof-of-concept experiments to show that lentiviral transduction of BacNav into cultured neonatal rat ventricular myocytes (NRVMs) can yield robust expression of functional channels and significantly augment cardiac action potential conduction via increase in peak Na+ current. Moreover, in vitro BacNav gene therapy in fibrotic NRVM cell cultures reduced occurrence of conduction block and reentrant arrhythmias. Furthermore, we showed that functional BacNav channels can be stably expressed in healthy mouse hearts six weeks following their delivery by intravenous injection of self-complementary AAV (scAAV) vectors coding BacNav gene, which in turn caused no adverse effects on cardiac electrophysiology. These results collectively demonstrated that BacNav channels can be directly, specifically, and stably expressed in CMs through viral gene delivery to augment myocardial excitability and conduction. In addition to enhanced peak Na+ current amplitude and CM excitability, based on the principles of cardiac excitation-contraction coupling, we assessed the ability of BacNav expression to also augment Ca2+ transient amplitude and contractility of CMs, as a potential two-pronged gene therapy strategy for treatment of heart failure. In multi-species studies in vitro and ex vivo, we showed that expression of BacNav enhanced Ca2+ transient amplitude and contractility of neonatal rat, mouse, and human CMs in a dose-depend manner by modulating the activity of the Na+/Ca2+ exchanger and increasing sarcoplasmic reticulum Ca2+ stores. This mechanism of BacNav action was further corroborated in silico, using a computational model of rabbit cardiac action potential. Importantly, to further support translational potential of in vivo BacNav therapy, we showed that AAV9-mediated BacNav expression rescued contractile deficit and prevented arrhythmogenicity in the settings of chronic cardiac pressure-overload in mice and acute myocardial infarction in non-human primates (NHPs). Furthermore, we established the safety of systemic and intramyocardial delivery of AAV9-BacNav in mice and NHPs, demonstrating the promise of BacNav gene delivery as a novel therapy for heart failure. Finally, we functionally screened previously uncharacterized BacNav variants, aiming to expand the therapeutic BacNav pool with novel channel candidates exhibiting faster gating kinetics that better resembles kinetics of mammalian cardiac Nav channels. One of the identified BacNav candidates from this screen, NavRhi, demonstrated improved gating kinetics and gain-of-excitability properties compared to our previously characterized NavSheP channel. We further optimized the membrane expression of NavRhi by comparing different human codon-optimized sequences, with additional enhancements achieved by creating a multicistronic vector incorporating two copies of NavRhi. By incorporating an Ankyrin G binding motif at the channel C-terminus, we also improved trafficking of NavRhi to the lateral membrane and intercalated disk, with the potential to further enhance cardiac conduction in vivo. In summary, this dissertation presents a novel therapeutic strategy to genetically augment cardiac conduction and contraction by revealing a unique dual-action mechanism of BacNav expression in cardiomyocytes. The encouraging in vivo results using diverse heart failure etiologies (pressure overload, myocardial infarction), animal models (mouse, NHP) and gene delivery routes (intravenous, intramyocardial) pave the way for future clinical translation of BacNav-based therapies for the treatment of heart disease. Moreover, this work establishes a comprehensive multi-species preclinical platform which will be instrumental in designing and validating innovative therapeutic strategies for heart failure and conduction disorders, offering a translational pipeline towards future clinical applications.