Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

Assessing Recombinant AAV Shedding After Cardiac Gene Therapy.

Gene therapy based on recombinant adeno-associated viral (rAAV) vectors has recently made significant progress as a clinical therapeutic. Unlike most traditional medications, gene therapy vectors can be biologically active when shed into the surrounding environment. Here we describe methods for collection and storage of multiple biological specimen samples and a PCR-based method for detection of shed adeno-associated viral (AAV) particles. We also describe a method for use of an infectious replication assay utilizing a cell line stably expressing AAV Rep and Cap genes and superinfection with adenovirus 5 to detect functionality in shed AAV particles.

Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method.

Gene therapy is making significant impact on a modest, yet growing, number of human diseases. Justifiably, the preferred viral vector for clinical use is that based on recombinant adeno-associated virus (rAAV). There is a need to scale up rAAV vector production with the transition from pre-clinical models to human application. Standard production methods based on the adherent cell type (HEK293) are limited in scalability and other methods, such as those based on the baculovirus and non-adherent insect cell (Sf9) system, have been pursued as an alternative to increase rAAV production. In this study, we compare these two production methods for cardiotropic rAAVs. Transduction efficiency for both production methods was assessed in primary cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and in mice following systemic delivery. We found that the rAAV produced by the traditional HEK293 method out-performed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This finding provides a potential caveat for vector function when using the baculovirus/Sf9 production system and underscores the need for thorough assessment of vector performance when using diverse rAAV production methods.

Gene and Cell Therapy for Cardiac Arrhythmias.

PURPOSE: In the last decade, interest in gene therapy as a therapeutic technology has increased, largely driven by an exciting yet modest number of successful applications for monogenic diseases. Setbacks in the use of gene therapy for cardiac disease have motivated efforts to develop vectors with enhanced tropism for the heart and more efficient delivery methods. Although monogenic diseases are the logical target, cardiac arrhythmias represent a group of conditions amenable to gene therapy because of focal targets (biological pacemakers, nodal conduction, or stem cell-related arrhythmias) or bystander effects on cells not directly transduced because of electrical coupling. METHODS: This review provides a contemporary narrative of the field of gene therapy for experimental cardiac arrhythmias, including those associated with stem cell transplant. Recent articles published in the English language and available through the PubMed database and other prominent literature are discussed. FINDINGS: The promise of gene therapy has been realized for a handful of monogenic diseases and is actively being pursued for cardiac applications in preclinical models. With improved vectors, it is likely that cardiac disease will also benefit from this technology. Cardiac arrhythmias, whether inherited or acquired, are a group of conditions with a potentially lower threshold for phenotypic correction and as such hold unique potential as targets for cardiac gene therapy. IMPLICATIONS: There has been a proliferation of research on the potential of gene therapy for cardiac arrhythmias. This body of investigation forms a strong basis on which further developments, particularly with viral vectors, are likely to help this technology progress along its translational trajectory.

Development of a sheep model of atrioventricular block for the application of novel therapies.

INTRODUCTION: Sheep have been adopted as a pre-clinical large animal for scientific research as they are good models of cardiac anatomy and physiology, and allow for investigation of pathophysiological processes which occur in the large mammalian heart. There is, however, no defined model of atrioventricular block in sheep to allow for pre-clinical assessment of new cardiac treatment options. We therefore aimed to develop an adult sheep model of atrioventricular block with the focus on future novel applications. METHODS AND RESULTS: We utilized six sheep to undergo two procedures each. The first procedure involved implantation of a single chamber pacemaker into the right ventricular apex, for baseline assessment over four weeks. The second procedure involved creating atrioventricular block by radiofrequency ablation of the His bundle, before holding for a further four weeks. Interrogation of pacemakers and electrocardiograms determined the persistence of atrioventricular block during the follow up period. Pacemakers were inserted, and atrioventricular block created in 6 animals using a conventional approach. One animal died following ablation of the His bundle, due to procedural complications. Four unablated sheep were assessed for baseline data over four weeks and showed 5.53 ± 1.28% pacing reliance. Five sheep were assessed over four weeks following His bundle ablation and showed continuous (98.89 ± 0.81%) ventricular pacing attributable to persistent atrioventricular block, with no major complications. CONCLUSION: We have successfully developed, characterized and validated a large animal model of atrioventricular block that is stable and technically feasible in adult sheep. This model will allow for the advancement of novel therapies, including the development of cell and gene-based therapies.

Analysis of recombinant adeno-associated viral vector shedding in sheep following intracoronary delivery.

Differences between mouse and human hearts pose a significant limitation to the value of small animal models when predicting vector behavior following recombinant adeno-associated viral (rAAV) vector-mediated cardiac gene therapy. Hence, sheep have been adopted as a preclinical animal, as they better model the anatomy and cardiac physiological processes of humans. There is, however, no comprehensive data on the shedding profile of rAAV in sheep following intracoronary delivery, so as to understand biosafety risks in future preclinical and clinical applications. In this study, sheep received intracoronary delivery of rAAV serotypes 2/6 (2 × 1012 vg), 2/8, and 2/9 (1 × 1013 vg) at doses previously administered in preclinical and clinical trials. This was followed by assessment over 96 h to examine vector shedding in urine, feces, nasal mucus, and saliva samples. Vector genomes were detected via real-time quantitative PCR in urine and feces up to 48 and 72 h post vector delivery, respectively. Of these results, functional vector particles were only detected via a highly sensitive infectious replication assay in feces samples up to 48 h following vector delivery. We conclude that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles for all excreta samples, except in the case of feces, where functional vector particles are present up to 48 h following vector delivery. These results may be used to inform containment and decontamination guidelines for large animal dealings, and to understand the biosafety risks associated with future preclinical and clinical uses of rAAV.

Enhanced cardiac repair by telomerase reverse transcriptase over-expression in human cardiac mesenchymal stromal cells.

We have previously reported a subpopulation of mesenchymal stromal cells (MSCs) within the platelet-derived growth factor receptor-alpha (PDGFRα)/CD90 co-expressing cardiac interstitial and adventitial cell fraction. Here we further characterise PDGFRα/CD90-expressing cardiac MSCs (PDGFRα + cMSCs) and use human telomerase reverse transcriptase (hTERT) over-expression to increase cMSCs ability to repair the heart after induced myocardial infarction. hTERT over-expression in PDGFRα + cardiac MSCs (hTERT + PDGFRα + cMSCs) modulates cell differentiation, proliferation, survival and angiogenesis related genes. In vivo, transplantation of hTERT + PDGFRα + cMSCs in athymic rats significantly increased left ventricular function, reduced scar size, increased angiogenesis and proliferation of both cardiomyocyte and non-myocyte cell fractions four weeks after myocardial infarction. In contrast, transplantation of mutant hTERT + PDGFRα + cMSCs (which generate catalytically-inactive telomerase) failed to replicate this cardiac functional improvement, indicating a telomerase-dependent mechanism. There was no hTERT + PDGFRα + cMSCs engraftment 14 days after transplantation indicating functional improvement occurred by paracrine mechanisms. Mass spectrometry on hTERT + PDGFRα + cMSCs conditioned media showed increased proteins associated with matrix modulation, angiogenesis, cell proliferation/survival/adhesion and innate immunity function. Our study shows that hTERT can activate pro-regenerative signalling within PDGFRα + cMSCs and enhance cardiac repair after myocardial infarction. An increased understanding of hTERT's role in mesenchymal stromal cells from various organs will favourably impact clinical regenerative and anti-cancer therapies.

Gene Therapy Approaches to Biological Pacemakers.

Bradycardia arising from pacemaker dysfunction can be debilitating and life threatening. Electronic pacemakers serve as effective treatment options for pacemaker dysfunction. They however present their own limitations and complications. This has motivated research into discovering more effective and innovative ways to treat pacemaker dysfunction. Gene therapy is being explored for its potential to treat various cardiac conditions including cardiac arrhythmias. Gene transfer vectors with increasing transduction efficiency and biosafety have been developed and trialed for cardiovascular disease treatment. With an improved understanding of the molecular mechanisms driving pacemaker development, several gene therapy targets have been identified to generate the phenotypic changes required to correct pacemaker dysfunction. This review will discuss the gene therapy vectors in use today along with methods for their delivery. Furthermore, it will evaluate several gene therapy strategies attempting to restore biological pacing, having the potential to emerge as viable therapies for pacemaker dysfunction.

Influence of Intramyocardial Adipose Tissue on the Accuracy of Endocardial Contact Mapping of the Chronic Myocardial Infarction Substrate.

BACKGROUND: Recent studies have demonstrated that intramyocardial adipose tissue (IMAT) may contribute to ventricular electrophysiological remodeling in patients with chronic myocardial infarction. Using an ovine model of myocardial infarction, we aimed to determine the influence of IMAT on scar tissue identification during endocardial contact mapping and optimal voltage-based mapping criteria for defining IMAT dense regions. METHOD AND RESULTS: In 7 sheep, left ventricular endocardial and transmural mapping was performed 84 weeks (15-111 weeks) post-myocardial infarction. Spearman rank correlation coefficient was used to assess the relationship between endocardial contact electrogram amplitude and histological composition of myocardium. Receiver operator characteristic curves were used to derive optimal electrogram thresholds for IMAT delineation during endocardial mapping and to describe the use of endocardial mapping for delineation of IMAT dense regions within scar. Endocardial electrogram amplitude correlated significantly with IMAT (unipolar r=-0.48±0.12, P<0.001; bipolar r=-0.45±0.22, P=0.04) but not collagen (unipolar r=-0.36±0.24, P=0.13; bipolar r=-0.43±0.31, P=0.16). IMAT dense regions of myocardium reliably identified using endocardial mapping with thresholds of <3.7 and <0.6 mV, respectively, for unipolar, bipolar, and combined modalities (single modality area under the curve=0.80, P<0.001; combined modality area under the curve=0.84, P<0.001). Unipolar mapping using optimal thresholding remained significantly reliable (area under the curve=0.76, P<0.001) during mapping of IMAT, confined to putative scar border zones (bipolar amplitude, 0.5-1.5 mV). CONCLUSIONS: These novel findings enhance our understanding of the confounding influence of IMAT on endocardial scar mapping. Combined bipolar and unipolar voltage mapping using optimal thresholds may be useful for delineating IMAT dense regions of myocardium, in postinfarct cardiomyopathy.

Therapeutic Prospects of Gene Therapy for Atrial Fibrillation.

Atrial Fibrillation (AF) is one of the most common types of cardiac arrhythmias experienced in clinical practice, increasing the risk of stroke, dementia, myocardial infarction and death. Currently available options for the treatment of AF use either pharmacological agents or catheter-based ablation therapies to restore sinus rhythm or control the ventricular response rate. These current treatment options are suboptimal at best, motivating research into discovering more effective and innovative ways to treat AF. Gene therapy is being explored for its potential to treat various human conditions including cardiac arrhythmias. Gene transfer vectors with increasing transduction efficiency and biosafety have been developed and trialled for cardiovascular disease treatment. With an improved understanding of the molecular mechanisms of AF, several gene therapy targets have been identified and evaluated in an attempt to rate or rhythm control the heart during AF. This review will discuss the gene therapy vectors in use today and methods for delivery of these vectors to the atrium. Further, it will evaluate several gene therapy strategies and approaches for sinus rhythm restoration and ventricular rate control that have the potential to emerge as a therapy for AF.

Observations on Attenuation of Local Electrogram Amplitude and Circuit Impedance During Atrial Radiofrequency Ablation: An In vivo Investigation Using a Novel Direct Endocardial Visualization Catheter.

AIMS: To define the temporal characteristics of atrial lesion growth (lesion surface area), local electrogram amplitude attenuation, and circuit impedance decrement during in vivo radiofrequency (RF) ablation with direct endocardial visualization (DEV). METHODS AND RESULTS: A direct endocardial visualization catheter was used for real-time endoscopic visualization of atrial endocardial surface during RF ablation. Videos of lesion growth (surface area), circuit impedance, and local electrogram amplitude were recorded during ablation in 11 ovine. Fifty-two atrial ablations at 12 W, 14 W, and 16 W power for 30 seconds were analyzed. During 30-second RF ablation, the lesion matured (90% of final lesion dimension) in the first 23.0 ± 5.8 seconds. The local electrogram amplitude attenuation (80% decrement) and circuit impedance attenuation (20% decrement from initial) occurred 13.8 ± 8.2 seconds and 13.1 ± 7.9 seconds, respectively, before lesion maturity in a significant proportion of 30 second atrial ablations. CONCLUSION: The DEV observations suggest that in smooth atrial surface ablations with significant local electrogram and impedance decrement in the first 10 seconds, further extension of ablation for 10-15 seconds could deliver optimal surface dimensions; however, real-time measurement of depth was not possible.