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Unlocking Precision Gene Therapy: Harnessing AAV Tropism with Nanobody Swapping at Capsid Hotspots

Hoffmann, M. D.; Gallant, J. P.; LeBeau, A. M.; Schmidt, D.

2024-03-27 synthetic biology
10.1101/2024.03.27.587049 bioRxiv
Show abstract

Adeno-associated virus has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2-fold valley and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. Our study shows that nanobody swapping at multiple capsid location is a viable strategy for nanobody-directed cell-specific AAV targeting.

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