AAV-mediated gene delivery to the cardiac conduction system

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO open technology program Biopace #18485 Background Gene therapy for cardiac arrhythmias has so far mostly focused on modifying the arrhythmogenic substrate in working myocardium or on creating biological pacemakers with viral vectors that did not selectively target the cardiac conduction system (CCS). As a result, CCS targeting by viral vectors is a relatively unexplored area. Aim In the current project, we explored the basic requirements for CCS targeting using adeno-associated virus (AAV) vectors. Methods Regular AAV9 and AAV-Myo4A capsid-pseudotyped AAV2 vectors (hereafter called AAV2/9 and AAV2/Myo4A vectors, respectively) expressing enhanced green fluorescent protein (eGFP) from a CMV or TNNT2 promoter, respectively, and an AAV2/9 vector expressing eGFP from a human GJA5 regulatory element were systemically injected in mice. After 4 weeks, hearts were harvested, fixed with paraformaldehyde and stained for TNNT2, eGFP, HCN4 and DNA to determine CCS transduction. Results Transduction of the sinoatrial and atrioventricular (AV) node using AAV2/9 vectors was very poor, while transduction was visible in the atrioventricular (i.e. His) bundle and working myocardium. In contrast, the transduction rate of the AV node and bundle using AAV2/Myo4A vectors were similar to that of the working myocardium. The human GJA5 regulatory element was able to restrict eGFP expression by AAV2/9 vectors to the AV bundle in 2/3 animals but the transduction efficiency was low. A third animal showed no transduction of any tissue, either indicative of the low transduction efficiency or of a failed injection. Conclusion This study not only provides proof-of-principle for CCS-targeted gene therapy but also shows that CCS transduction can be improved through AAV capsid engineering and be restricted to (parts of) the CCS by using specific transcriptional control elements.