PDF(Pubmed)
Abstract：
Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.
摘要：
尽管对AAV转基因释放的分子理解仍然有限,但腺相关病毒(AAV)作为基因治疗的递送载体正在获得关注。通常,病毒脱衣的过程是通过热应激（体外）进行研究的，揭示衣壳在高温下崩解。为了评估不同空的和填充的AAV制剂的稳定性，我们使用了基于光散射的干涉显微镜技术的质量测光，在单粒子的基础上，确定AAV的分子量。通过引入热稳定的DNA质粒作为内标，我们定量探讨了热量对AAV的影响。一般来说,空AAV表现出比基因组填充颗粒更大的耐热性。我们的数据还表明，在DNA释放后，衣壳不会变成空的AAV，但似乎聚集或瓦解。引人注目的是，一些AAV表现出中间状态，衣壳被破坏，但结合的基因组被保留，与核酸酶孵育后才在实验上出现的特征。我们的数据表明，热脱涂层过程是高度AAV特异性的(即，会受到血清型的影响，基因组,主机系统)。我们认为核酸酶处理与MP组合可用作评估重组和/或临床AAV载体结构完整性的额外分析工具。
