• Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2.

Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2.

Molecular therapy. Methods & clinical development (2020-06-04)
Marti Cabanes-Creus, Adrian Westhaus, Renina Gale Navarro, Grober Baltazar, Erhua Zhu, Anais K Amaya, Sophia H Y Liao, Suzanne Scott, Erwan Sallard, Kimberley L Dilworth, Arkadiusz Rybicki, Matthieu Drouyer, Claus V Hallwirth, Antonette Bennett, Giorgia Santilli, Adrian J Thrasher, Mavis Agbandje-McKenna, Ian E Alexander, Leszek Lisowski

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.

Product Number
Product Description

Trimethoprim, ≥98.5%
Penicillin-Streptomycin, Solution Stabilized, with 5,000 units penicillin and 5mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Hanks′ Balanced Salt solution, Modified, with sodium bicarbonate, without calcium chloride and magnesium sulfate, liquid, sterile-filtered, suitable for cell culture
Heparin sodium salt from porcine intestinal mucosa, Grade I-A, ≥180 USP units/mg, powder, BioReagent, suitable for cell culture
Fetal Bovine Serum, Australia origin, suitable for, USDA approved, sterile-filtered, suitable for cell culture, suitable for hybridoma