Functional evaluation of a natural AAV capsid liver targeting motif in human hepatocytes

Unzu, C.; Chen, A. X.; Mancio-Silva, L.; Zinn, E.; Wen, Y.; Llinares, C.; LLanos, A.; Zhu, C.; Fieldsend, A.; Sanmiguel, J.; Bissig-Choisat, B.; Bissig, K.-D.; Alexander, I.; Bhatia, S.; Vandenberghe, L. H.

doi:10.64898/2026.08.20.745184

bioRxiv · 10.64898/2026.08.20.745184

Functional evaluation of a natural AAV capsid liver targeting motif in human hepatocytes

Unzu, C.·Chen, A. X.·Mancio-Silva, L.·Zinn, E.·Wen, Y.·Llinares, C.·LLanos, A.·Zhu, C.·Fieldsend, A.·Sanmiguel, J.·Bissig-Choisat, B.·Bissig, K.-D.·Alexander, I.·Bhatia, S.·Vandenberghe, L. H.

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Abstract

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

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Unzu, C., Chen, A. X., Mancio-Silva, L., Zinn, E., Wen, Y., Llinares, C., LLanos, A., Zhu, C., Fieldsend, A., Sanmiguel, J., Bissig-Choisat, B., Bissig, K.-D., Alexander, I., Bhatia, S., Vandenberghe, L. H.. 2026-08-20. Functional evaluation of a natural AAV capsid liver targeting motif in human hepatocytes. https://doi.org/10.64898/2026.08.20.745184

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