Intramyocardial Tbx18 Gene Delivery In A Radiopaque Medium Enables Real-Time Assessment Of Gene Transfer, And Is Compatible With Biological Pacemaker Function

Introduction: The efficacy and specificity of myocardial gene transfer depends on retention of the injectate at the site of gene delivery. However, no direct method exists to confirm myocardial retention of a biologic delivered via an intracardiac transvenous catheter. We tested feasibility of a radiographic contrast media as a gene delivery vehicle, and fluoroscopic assessment of intramyocardial retention as well as proper function of a transgene. Methods and Results: Synthetic mRNA expressing GFP in saline was mixed with an iodine-based radiopaque contrast agent, iopamidol, at 2, 5, 20, 30 and 40%. The minimum content of iopamidol that allowed unequivocal radiographic identification ex vivo was 20%. To validate functional expression of the transgene, GFP mRNA was injected focally into the left ventricular apex of the rat heart upon thoracotomy at 0, 2, 5 or 20% iopamidol solution. On day 5, the hearts were harvested and examined for GFP expression, which illustrated comparable and focal GFP expression in all animals. To test whether gene delivery in a contrast media is compatible with intended disease-modifying activity, we employed TBX18 gene transfer via the right femoral vein in a porcine model of complete heart block. We have previously demonstrated that TBX18 could convert ventricular myocytes to pacemaker cells, and create de novo ventricular pacing in situ. Intracardiac delivery of TBX18 or GFP (n=6 or 2 pigs, respectively) mRNA dissolved in 20% iopamidol to the high His bundle region revealed clear, real-time visualization of the injectate in all pigs. In one of the 8 pigs, the injectate diffused rapidly into the ventricular circulation, which necessitated a repeat injection. The maximum heart rate of TBX18-injected pigs was faster than that of GFP-injected pigs upon β-adrenergic stimulation at 77±12 vs. 43±13 BPM at week 2 ( P =0.08), and at 93±19 vs. 48±3BPM at week 4 ( P <0.05), respectively. Conclusion: Radiographic contrast agent can serve as an effective gene delivery vehicle, allowing real-time and non-invasive assessment of the retention of intramyocardial gene delivery. This approach may be generalizable to other focal gene therapy applications, and minimize suboptimal gene delivery.