AAV6-mediated gene transfer of HCN1-ddd generates slightly faster baseline beating rates as compared to Hcn2 yet with a potential risk for pro-arrhythmia

Abstract Electronic pacing is the current treatment of choice for complete heart block (CHB) that revolutionised last century’s standard of care. Despite its success, some critical issues remain such as providing suboptimal cardiac output and a lack of direct autonomic responsiveness. To develop a more physiological and hardware-free pacemaker, we focused on developing gene therapy-based biological pacemakers by means of ion channel over-expression. Previous studies with adenoviral gene transfer indicated possible superior outcomes for the engineered mutant HCN1-ΔΔΔ as compared to wild-type Hcn2, yet a head-to-head comparison was never made. To assess the long-term biological pacemaker efficacy for AAV6-mediated gene transfer of HCN1-ΔΔΔ and Hcn2. Functional biological pacemaker studies were conducted in a porcine model of radiofrequency ablation-induced CHB. Four weeks after the ablation, animals were studied in two different groups: AAV6-cTnT-HCN1-ΔΔΔ and AAV6-cTnT-Hcn2, both immunosuppressed with prednisolone and Cyclosporine A. All animals were then followed for another four weeks to evaluate in vivo biological pacemaker performance and then organs were harvested to assess transduction efficiency. Biological pacing effectiveness was evaluated in view of maximal (max) and basal (mean) beating rates (HR), and pacemaker dependency that animals had shown throughout the study. Four weeks after the induction of CHB, on the day of the injection, all animals (n = 6) presented an average mean HR of 53,67 ± 7,64 bpm (Figure 1B) and a pacemaker dependency above 65% (Figure 1C). One week after gene transfer, these beating rates rapidly increased and pacemaker dependency declined. Hcn2 and HCN1-ΔΔΔ groups displayed a notable increase in max HR (Figure 1A), where HCN1-ΔΔΔ offered the most potent and sustained response (Hcn2 153,33 ± 47,26 bpm vs. HCN1-ΔΔΔ 220 ± 43,59 bpm; Figure 1A). In terms of mean HR, they similarly trended up in both groups (Hcn2 57,81 ± 5,49 bpm vs. HCN1-ΔΔΔ 59,22 ± 2,28 bpm; Figures 1B and E). Lastly, these faster beating rates in HCN1-ΔΔΔ resulted in modestly lower dependency in electronic back-up pacing (52 ± 7,94 % vs. 48,3 ± 22,37 % for HCN1-ΔΔΔ vs. HCN2; Figures C and F). AAV6-mediated HCN1-ΔΔΔ gene transfer produced slightly faster mean heart rates as compared to Hcn2, yet HCN1-ΔΔΔ generated persistently increased maximal heart rates (in two out of three animals) that exceeded the physiological desirable range. These findings possibly relate to the much faster channel gating kinetics of HCN1-ΔΔΔ that were previously found to be profoundly pro-arrhythmic with the chimera channel HCN212.Figure 1Abstract ESC 2023