Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

中文翻译：

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多重AAV库中的交叉包装和衣壳马赛克形成。

腺相关病毒（AAV）变体的文库的产生和筛选已成为鉴定用于基因治疗应用的新型衣壳的有效方法。对于大多数文库，巨大的种群多样性需要多重生产，其中将含有反向末端重复序列（ITR）的质粒变体的文库一起转染到细胞中以生成病毒文库。此过程有可能被交叉包装和镶嵌技术混淆，其中颗粒由基因组和衍生自不同文库成员的衣壳单体组成。在这里，我们使用旨在评估衣壳成分和包装保真度的新颖检测方法，在简化的最小文库中研究了交叉包装和镶嵌的普遍性。我们显示，AAV文库变体在高质粒水平下产生时易于交叉包装和衣壳镶嵌形成，尽管程度不及重组环境。我们还提供了实验证据，即稀释输入文库DNA会显着提高衣壳单体的同质性，并增加AAV文库中的衣壳：基因组相关性。最后，我们确定当用于体内筛选时，类似的稀释方法会产生更高质量的文库。在一起，这些发现定量地描述了AAV库中交叉包装和镶嵌的流行，并确定了在后续屏幕中将相关噪声降至最低的条件。我们还提供了实验证据，即稀释输入文库DNA会显着增加衣壳单体的同质性，并增加AAV文库中的衣壳：基因组相关性。最后，我们确定当用于体内筛选时，类似的稀释方法会产生更高质量的文库。在一起，这些发现定量地描述了AAV库中交叉包装和镶嵌的流行，并确定了在后续屏幕中将相关噪声降至最低的条件。我们还提供了实验证据，即稀释输入文库DNA会显着增加衣壳单体的同质性，并增加AAV文库中的衣壳：基因组相关性。最后，我们确定当用于体内筛选时，类似的稀释方法会产生更高质量的文库。在一起，这些发现定量地描述了AAV库中交叉包装和镶嵌的流行，并确定了在后续屏幕中将相关噪声降至最低的条件。