Dichotomies in Ex Vivo and In Vivo Performance of Receptor-Binding Mutants of Adeno-Associated Virus Vectors

Abstract

Receptor engagement plays a key role in the cellular uptake, intracellular trafficking, and overall transduction efficiency of adeno-associated virus (AAV) vectors. Although heparan sulfate proteoglycan (HSPG) and α5ß1 integrin-binding motifs of the AAV serotype 2 (AAV2) capsid were mapped, it has remained incompletely understood how loss of these interactions affects AAV vector performance. Hence, we generated capsid variants harboring mutations at capsid residues responsible for HSPG binding (AAV2ΔHSPG), α5ß1 integrin binding (AAV2ΔIntegrin), or both (AAV2ΔHSPGΔIntegrin), and investigated the mutants systematically ex vivo and in vivo. While neither production nor packaging efficiency was affected, variants revealed distinct physicochemical alterations, including altered electrophoretic mobility and thermal stability. Ex vivo, loss of HSPG binding completely abolished transgene expression across various cell lines, whereas ablation of α5ß1 integrin binding lowered transduction efficiency. While cellular uptake was reduced for AAV2ΔIntegrin and almost eliminated for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin, mutants nevertheless reached the nuclear compartment, albeit with lower efficiency compared with AAV2. Strikingly, in vivo performance diverged sharply from ex vivo findings as we observed a substantially enhanced transduction in multiple non-hepatic tissues for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin in C57BL/6N albino and BALB/c mice, as well as a strong liver detargeting, while AAV2ΔIntegrin was non-infectious in vivo. These data uncover a fundamental dichotomy between ex vivo and in vivo determinants of AAV2 transduction and identify receptor-binding ablation, especially integrin binding, as a potential alternative to detarget AAV2 vectors for next-generation capsid engineering and tissue-specific retargeting.