Zeng Cheng, Moller-Tank Sven, Asokan Aravind, Dragnea Bogdan
Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.
J Phys Chem B. 2017 Mar 2;121(8):1843-1853. doi: 10.1021/acs.jpcb.6b10131. Epub 2017 Feb 14.
Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid-genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed.
重组腺相关病毒（AAV）是一种很有前景的基因治疗载体。为了在这个方向上取得进展，必须详细了解基因组货物的特性与衣壳稳定性之间的关系。本研究的目的是确定包装的载体基因组在AAV颗粒对机械压缩的响应以及与底物的粘附方面所起的作用。具体而言，我们使用原子力显微镜比较了空的2型腺相关病毒（AAV2）衣壳与包装单链DNA或自互补DNA的AAV2载体的力学性能。我们发现，所有类型的病毒在从缓冲液吸附到原子级平整的石墨表面时都会发生部分变形。吸附时，观察到衣壳相对于底物朝着二重对称轴有一个优先取向。这种偏向的程度取决于货物类型，表明界面性质可能受货物影响。所有颗粒在破裂前都表现出显著的相对应变。与明显依赖货物的界面相互作用不同，对定向应力的弹性响应在很大程度上由衣壳性质主导。然而，装载不同货物的颗粒之间存在可测量的微小差异；自互补AAV（scAAV）载体对外部压缩最具弹性。我们还展示了如何根据多变量条件概率方法分析弹性常数和破裂力数据集，以便基于从纳米压痕试验获得的力学性能数据库来确定基因组含量。本文讨论了对理解重组AAV衣壳 - 基因组相互作用如何影响载体稳定性和基因治疗应用有效性的意义。
