Use of the prototypical AAV2 capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for haemophilia 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 eleven amino acids 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 two of the eleven 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 varaints, N496D and N582S, which are highly functional in vivo. Both substitutions 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.

使用原型AAV2衣壳在乙型血友病早期肝靶向基因治疗试验中出乎意料的适度功效。这一结果与在嵌合小鼠-人肝脏中产生的后续数据一致，表明AAV2衣壳在体内转导了原代人肝细胞效率低下。相反，在同一模型中通过定向进化产生的新变体，例如AAV-NP59，可以高效地转导原代人肝细胞。尽管这些经验数据具有巨大的翻译意义，但尚未充分阐明支持这种增强的原代人肝细胞AAV衣壳转导性能的机制。值得注意的是，AAV-NP59与原型AAV2衣壳仅11个氨基酸不同，可作为研究衣壳序列/结构与载体功能之间相关性的工具。

使用两种正交矢量方法，我们已经确定，AAV-NP59（T503A和N596D）中存在的11种变化中，只有2种是这种衣壳变异体在人原代肝细胞体内的转导性能增强的原因，我们已经将其与硫酸乙酰肝素蛋白聚糖（HSPG）的结合亲和力减弱。为了支持该假设，我们使用定向进化方法鉴定了两个另外的单个氨基酸取代AAV2变异蛋白N496D和N582S，它们在体内具有较高的功能。两种取代突变均降低了AAV2对HSPG的亲和力。最后，我们调节了高度鼠肝型血清型AAV8与HSPG相互作用的能力。结果支持我们的假设，即增强的HSPG结合力可能会对其他强烈肝变种的体内功能产生负面影响，并且与HSPG相互作用的调节对于确保体内最大效率至关重要。通过这项研究获得的见解可能对针对肝和其他器官的临床前和临床应用的AAV生物学和衣壳发育研究产生重大影响。