Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.

中文翻译：

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在单衣壳水平表征 AAV 载体颗粒稳定性

病毒家族进化出不同的基因组脱壳策略，然后是重组载体。源自腺相关病毒 (AAV) 的载体被认为是体内基因转移的主要递送工具，尤其是基因治疗。我们结合使用原子力显微镜 (AFM)、生化实验和物理建模，在这里研究了 AAV 载体粒子的物理特性和稳定性。我们首先比较了源自两种不同血清型（AAV8 和 AAV9）的 AAV 载体的形态学特性。此外，我们通过将载体颗粒孵化到增加受控温度来触发 ssDNA 脱壳。我们在单粒子水平上进行的分析表明，基因组释放可以通过两种替代途径在体外发生：要么衣壳保持完整并线性地排出 ssDNA 分子，要么衣壳破裂，使 ssDNA 保持紧密的缠结构象。对喷射基因组长度分布的分析进一步揭示了两步喷射行为。我们提出了一个动力学模型，旨在定量描述衣壳和基因组沿不同途径的进化，作为时间和温度的函数。该模型允许量化 AAV8 和 AAV9 粒子的相对稳定性。作为时间和温度的函数。该模型允许量化 AAV8 和 AAV9 粒子的相对稳定性。作为时间和温度的函数。该模型允许量化 AAV8 和 AAV9 粒子的相对稳定性。