In vivo promoter biopanning with AAV

Recombinant Adeno-associated viruses (AAV) are flagship vectors for in vivo human gene therapy. An integral vector element are promoters, which control transgene expression in either a ubiquitous or cell-type-selective manner. Identifying optimal capsid-promoter combinations is challenging, especially when considering on- versus off-target expression. Therefore, we established a pipeline for in vivo promoter biopanning in AAV that expands on our AAV capsid barcoding technology. Here, we illustrate its potential by screening 53 promoters in 16 murine tissues in AAV9, based on its broad tropism that enables comprehensive in vivo comparisons. Surprisingly, the 2.2 kb human GFAP (glial fibrillary acidic protein) promoter was the top hit in the liver, where it outperformed robust benchmarks such as the LP1 or human alpha-1-antitrypsin promoters. Analysis of hepatic cell populations revealed a preferred GFAP promoter activity in hepatocytes. Notably, GFAP also surpassed the LP1 and CMV promoters in human hepatocytes in a humanized mouse setting. Together, this makes the GFAP promoter an exciting candidate for applications requiring efficient and specific hepatic transgene expression. Our pipeline expands the arsenal of technologies for high-throughput in vivo screening of viral vector components and is compatible with capsid barcoding, facilitating the future combinatorial interrogation of complex AAV libraries.