Towards enhancing photostability and adeno-associated viral vector delivery for genetically encoded plasma fluorescent labels.

Significance: Investigating blood microcirculation is important to better understand cardiovascular, metabolic, and neurodegenerative diseases. Although visualization of microcirculation has traditionally relied on the injection of short-lived fluorescent tracers, this approach poses challenges for experiments in awake animals or long-term imaging. To address these limitations, we previously developed a genetically encoded fluorescent plasma label that enables stable, minimally invasive Aim: We aim to extend the utility of genetically encoded plasma labeling by testing brighter, more photostable fluorescent proteins and evaluating strategies to enhance adeno-associated viral (AAV)-mediated expression. Approach: AAV8 vectors encoding albumin fused to mNeonGreen or StayGold variants were administered systemically. Expression levels were tracked via blood sampling, and the effects of secondary AAV injection and immunomodulation were tested. Results: Alb-mStayGold showed improved photostability but lower brightness than Alb-mNeonGreen. Secondary intraperitoneal AAV delivery successfully induced transgene expression even after prior AAV exposure. Moreover, higher AAV8-induced plasma label expression in males is confirmed. Immunosuppressant treatment increased plasma fluorescence, whereas neonatal AAV injection failed to induce tolerance. Conclusion: Photostable plasma labeling and immune modulation strategies expand the applicability of AAV-based vascular imaging.