Directed Evolution of Adeno-associated Virus Targeting Heart and Skeletal muscle

The goal of this project is to engineer gene vectors that target a single tissue type, especially striated muscles. A random library of adeno-associated virus (AAV) was constructed by shuffling the capsid genes of AAV serotypes 1 to 9, and the resulting chimeric genes were screened for muscle specificity by direct in vivo panning. Viruses expressed high in muscles were collected, and the process was repeated to construct chimeric AAV library. To further improve specificity, in vitro biopanning was done on three types of cultured cells that represent kidney, liver and muscles. For the desirable features, selection was based on infection rate, preferably high in muscle but low in liver. Using AAV2 as a standard, three reconstructed AAV were found to be expressed at higher frequencies in myotubes with comparable or lower frequencies in hepatocytes. These recombined AAV would be further selected in vivo through tail vein injection in mice to identify the virus that was the most specific to muscles.