P03

A highly sensitive nanoluciferase reporter enables efficient assessment of functional and genomic biodistribution of AAV in mice

M Seirup(1) K Wingate(1) T Uyeda(3) I Prost(2) M Denhart(1) K Machleidt(1) D Horejsh(1) W Zhou(3) T Hoang(1) M Urh(1)

1:Promega Corp; 2:Promega France; 3:Promega Biosciences

Engineering of AAV capsids has been a favorable approach to enhance specificity toward target tissues while minimizing off-target delivery. AAV variant selection often comprise of large sample sets and a complex workflow that requires automation. Each animal is injected with a variant AAV capsid library, followed by the isolation, purification and sequencing of DNA from each AAV infected tissue. Herein we report a simple, fast and automated workflow to enable extraction, purification and quantification of viral DNA from tissue samples in a high throughput fashion. We generated AAV9 viruses expressing a NanoLuc® reporter to examine tissue tropism in animals via bioluminescent live imaging. Two weeks after injection, bright and dose dependent bioluminescent images were captured via live whole-body imaging. Next, tissues were collected, and total DNA was extracted and purified using the Maxwell® nucleic acid extraction system. Total DNA quantity and purity were assessed, followed by determination of AAV viral titers by normalization to house-keeping gene Tert using digital droplet PCR. We found AAV titers of several organs correlated with their luminescent signals. The viral titers accumulated in targeted tissues were also strongly dependent on the injection dose. In summary, we report a versatile NanoLuc® reporter that is bright and well suited to examine tissue tropism in live animals. In addition, we provide a simple, single day workflow which enables high throughput compatible viral titer determination of AAV infected tissues. The combination of our reporter and our proposed workflow would advance efficiency and effectiveness when screening AAV capsid libraries.