Description
Adeno-associated virus serotype 9 (AAV9) is a popular tool in cardiac research due to its cardiotropism, high transduction efficiency, and little to no pathogenicity, making it highly applicable for genetic manipulation, in vivo. However, current AAV9 technology is limited by the lack of chamber-specific strains that can selectively express and elucidate gene function in an atrial- and ventricular-specific manner. In fact, study of gene function in atria has been limited due to the lack of an appropriate tool to study atria specifically in vivo, hindering progress in the study of atrial-specific diseases such as atrial fibrillation, the most common cardiac arrhythmia in the U.S. Due to their robust and chamber-specific expression in mouse cardiac tissue across development and with pathology, it was hypothesized that Nppa and Myl2 promoters can be configured into recombinant forms of AAV9 so as to confer atrial- and ventricular- myocyte specific expression of desired transgenes, respectively. Promoter fragments of Nppa and Myl2 driving a reporter gene were tested in vitro and in an AAV9 vector in vivo to determine the regulatory elements necessary for robust, AAV9-mediated chamber-specific expression; these regulatory elements were determined to be confined within 450 and 262 base pairs of the Nppa and Myl2 promoters, respectively. A preliminary study assessing chamber-specific loss-of-function of a test gene, valosin-containing protein (VCP), was conducted using the Nppa and Myl2 driven AAV9 vectors to generate atrial- and ventricular-specific VCP knockout (KO) mice, which were then subjected to transverse aortic constriction (TAC). After 4 weeks of TAC, atrial- and ventricular-specific VCP KO produced differential phenotypes, demonstrating distinct gene function in atrial and ventricular myocytes. At only 450 and 262 base pairs of Nppa and Myl2 promoters, respectively, the chamber-specific AAV9 vectors address limitations to current AAV9 technology, enhancing chamber-specificity while accommodating insertion of transgenes as large as 4,000 base pairs.
