Introduction: Cardiac conduction system (CCS) dysfunction contributes significantly to morbidity and mortality associated with various pathologies, including heart failure. Evidence suggests that remodelling of CCS gene expression, particularly in ion channels regulating its function, are a leading cause of CCS dysfunction. Despite the emergence of these promising therapeutic targets, few methods are available to target the CCS for genetic modulation. Viral vectors offer a powerful method for gene delivery, and AAV9 has been shown to target the working myocardium efficiently. We aimed to utilise the cardiotropic AAV9 vector to modulate gene expression in the CCS from a single systemic intravenous dose, and spatially restrict transgene expression via tissue specific promoters. Doing so would provide a means of validating therapeutic targets of CCS disease, and offer a targeted means of correcting function. Methods: Firstly, tissue specific promoter candidates for use in a CCS specific gene therapy vector were characterised. Two genes where expression is restricted to the CCS in the adult mouse heart were selected; HCN4, a key pacemaking ion channel responsible for If, and KCNE1, which in combination with KVLQT1 regulates IK,s. We also selected the cardiac specific cTnT promoter and the ubiquitous CMV promoter as a positive control. Promoters were cloned upstream of GFP to provide quantitative information on their transcriptional activity. All promoters were first tested in vitro in a sinoatrial node-like ESC derived cell line (Shox2) to assess transcriptional strength, and in off-target cells including HEK293, HIH-3T3 to assess tissue specificity. To enhance transcriptional strength, the 380 bp CMV enhancer was appended upstream of each promoter. After in vitro validation, the most promising promoter constructs were packaged in AAV9 for in vivo testing. Results: All CCS tissue specific promoter candidates showed relatively high activity in Shox2 cells, and low activity in all off target cells. The CMV enhancer increased transcriptional strength modestly in Shox2 cells, with only the CMVe 0.8 kb KCNE1 construct reaching significance. Some evidence of promiscuous activity in HEK293 and NIH-3T3 cells was also noted with CMVe. AAV9-CMV induced high level transgene expression in all tissues examined in vivo, particularly the heart and liver. The SAN was effectively targeted by AAV9, with a transduction efficiency of 65% using AAV9-CMV. CCS specific promoter candidates conferred no detectable transgene expression in any tissues examined. AAV9-cTnT was highly active in the cardiac ventricles and atria, and achieved a transduction efficiency of 7.23% in the SAN. Conclusions: A single systemic intravenous injection of AAV9 is an effective means of targeting the SAN in vivo. Further work is required to elucidate the mechanisms surrounding fine patterning of gene expression in the CCS towards development of potent tissue specific promoter constructs.