Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2
Abstract
Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo , an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo . Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo . The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.
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BibTeX
@article{cabanescreus2026attenuation,
title = {{Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2}},
author = {Marti Cabanes-Creus and Adrian Westhaus and Renina Gale Navarro and Grober Baltazar and Erhua Zhu and Anais Amaya and Sophia Liao and Suzanne Scott and Erwan Sallard and Kimberley Dilworth and Arkadiusz Rybicki and Matthieu Drouyer and Claus Hallwirth and Antonette Bennett and Giorgia Santilli and Adrian Thrasher and Mavis Agbandje-Mckenna and Ian Alexander and Leszek Lisowski and Sophia H.Y. Liao},
journal = {Molecular Therapy - Methods and Clinical Development},
year = {2026},
month = sep,
doi = {10.1016/j.omtm.2020.05.004},
url = {https://hal.science/hal-05771971v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2
AU - Marti Cabanes-Creus
AU - Adrian Westhaus
AU - Renina Gale Navarro
AU - Grober Baltazar
AU - Erhua Zhu
AU - Anais Amaya
AU - Sophia Liao
AU - Suzanne Scott
AU - Erwan Sallard
AU - Kimberley Dilworth
AU - Arkadiusz Rybicki
AU - Matthieu Drouyer
AU - Claus Hallwirth
AU - Antonette Bennett
AU - Giorgia Santilli
AU - Adrian Thrasher
AU - Mavis Agbandje-Mckenna
AU - Ian Alexander
AU - Leszek Lisowski
AU - Sophia H.Y. Liao
PY - 2026
DA - 2026/09/30
JO - Molecular Therapy - Methods and Clinical Development
DO - 10.1016/j.omtm.2020.05.004
UR - https://hal.science/hal-05771971v1
AB - Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo , an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo . Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo . The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.
SN - 2329-0501
ER -