Evaluation of different rAAV serotypes and their tropism for the lung using non-invasive imaging in a fetal mouse model

Background: The fetal mouse lung is typically targeted by intra-amniotic (IA) injection. We evaluated the potential of intratracheal (IT) delivery by comparing IT with IA injection using fluorescent molecules. Next, we injected different rAAV serotypes (rAAV6.2, rAAV5 and rAAV9) encoding FLUC and b-gal to assess their efficacy targeting the fetal lung. BLI was combined with MRI to obtain a more accurate localization of gene expression in vivo. Methods: Fetuses of pregnant NMRI were injected IT or IA at E18 with 30 mL fluospheres (n ¼ 5) or rAAV (n ¼ 3 per serotype). Combined BLI-MRI of injected pups was performed at 1 week. For co-registration, BLI images were overlaid with whole-body MRI using newly developed software. Mice were sacrificed at 1 and 4 weeks. X-gal staining was performed on frozen sections to localize gene expression. Results: IT injection was more efficient in delivering fluospheres to the fetal lung compared to IA. BLI-MRI coregistration showed FLUC expression in the lung region after IT delivery of rAAV6.2, in the lung region and nose after rAAV5 and predominantly in the abdomen after rAAV9. Confirmation of transduction was obtained after X-gal staining on tissue sections. rAAV6.2 mainly targeted the lung, rAAV5 showed b-gal positive cells in lung and nose and rAAV9 targeted the lung and liver. Conclusions: We developed a novel fetal mouse model for lung transduction. Combining BLI with MRI allowed for in vivo evaluation of different rAAV serotypes and their tropism for the lung. In the future, this fetal mouse model can be used to investigate gene therapy for genetic disorders.status: publishe