The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
The Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Nat Commun. 2019 Jul 30;10(1):3415. doi: 10.1038/s41467-019-11321-7.
Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.
传统的腺相关病毒 (AAV) 载体转导模式识别方法基于报告基因的高、稳定表达。因此，传统描述的嗜性忽略了经历瞬时转导的细胞类型，或者具有低但无法检测到的报告基因表达水平。这为基于 AAV 的基因组编辑应用创造了一个盲点，因为活性只需要最小程度的转基因表达。在这里，我们使用编辑报告小鼠来填补这一空白。我们的方法灵敏地捕捉来自 AAV 载体的高和低转基因表达。使用 AAV8 和其他血清型，我们在与传统方法的并排比较中证明了该方法的优越性，证明了许多以前未知的 AAV 靶向部位，并更好地预测了 AAV-CRISPR 递送至基因编辑后留下的足迹。我们预计，这种在体内从 AAV 载体捕捉全谱转导模式的系统将成为当前和新兴的 AAV 技术的基础。
