Guo, Y.; Foiret, J.; Seo, J. W.; Zhang, N.; Wang, J.; Raie, M. N.; Jan, B. L.; Tumbale, S. K.; Ferrara, K.

Gene therapy using adeno-associated virus (AAV) vectors shows promise for cancer treatment through molecular intervention, yet achieving sufficient and targeted delivery to brain tumors via systemic administration remains limited by the biological barriers. Here, we investigate whether microbubble-enhanced focused ultrasound (MB-FUS) enhances targeted delivery of systemically administered AAV to orthotopic gliomas, using quantitative PET imaging of 64Cu-radiolabeled AAV9 vectors and fluorescent reporter expression to assess biodistribution and functional efficacy. PET imaging at 21 hours post-injection revealed 3.2-fold higher 64Cu-AAV accumulation in MB-FUS-treated tumors compared to non-sonicated tumors (n=3, p=0.004). Quantitative PCR analysis of tumor tissue at the same timepoint confirmed enhanced vector delivery, demonstrating 6.4-fold increased genome copies in MB-FUS-treated tumors (p=0.0003). Optical reporter gene imaging at 17 days post-treatment revealed that enhanced vector delivery translated to significantly increased transgene expression, with 5.3-fold higher transduction in MB-FUS-treated tumors (p=0.0002). These results establish that MB-FUS enables spatially-targeted AAV delivery with quantifiable enhancement of both acute vector biodistribution and downstream transgene expression. The integration of radiolabeled AAV with PET imaging provides a non-invasive methodology for real-time assessment of vector delivery and optimization of treatment protocol for brain cancer gene therapy.