Enhancing atrial specific gene expression using a calsequestrin cis-regulatory module 4 with a sarcolipin promoter.

BACKGROUND: Cardiac gene therapy using the adeno-associated virus serotype 9 vector is widely used due to its efficient transduction. However, the promoters used to drive expression often cause off-target localization. To overcome this, former studies have applied cardiac specific promoters but the expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial specific gene expression, an enhancer calsequestrin cis-regulatory module 4 (CRM4) and the highly atrial specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression.

METHODS: To observe expression and bio-distribution, constructs were generated using two different reporter genes: luciferase and EGFP. The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN), and the CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed by luciferase assay, western blot, qPCR, and fluorescent imaging.

RESULTS: The cytomegalovirus promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module sarcolipin combination showed enhanced atrial expression and minimized off target effect even when compared to the individual sarcolipin promoter.

CONCLUSION: For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra-atrial expression.