Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.

中文翻译：

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通过高通量测序追踪腺相关病毒衣壳进化。

尽管在临床基因治疗试验中使用重组腺相关病毒 (rAAV) 载体取得了早期成功，但仍然存在局限性，因此有必要进一步取得进展。一些挑战包括不同水平的预先存在的中和抗体和特定目标组织和/或疾病中的不良转导。此外，由于针对衣壳的免疫反应，通常不可能重新使用 rAAV 载体。具有新型衣壳的重组腺相关病毒 (AAV) 载体可以在自然界中分离或通过不同的定向进化策略开发。然而，在大多数情况下，AAV 选择的过程并没有得到很好的理解，需要新的策略来定义最佳参数，以开发更高效和功能更强大的 rAAV 衣壳。因此，对 AAV 衣壳库使用条形码，它可以通过高通量测序进行筛选，提供了一个强大的工具来跟踪 AAV 衣壳进化并可能改进 AAV 衣壳库筛选。在这项研究中，我们研究了不同的参数如何影响两种人类细胞类型中两个不同 AAV 文库的筛选。我们发现了关于如何最大化获得具有所需特性的 AAV 变体的可能性的新的和意想不到的见解。该研究的主要发现如下。(1) 包含用于 AAV 复制的辅助病毒可以选择性地传播可以复制到更高滴度的变体，但不一定在转导方面更好。(2) 具有特定衣壳的 AAV 之间的竞争可以发生在已经感染不同 AAV 的细胞中。(3) 使用低感染复数进行感染会导致屏幕之间的差异更大，并且在选择最需要的衣壳时不是最佳选择。(4) 采用多轮选择可能会适得其反。我们得出结论，在筛选 AAV 文库以增强感兴趣的特性时，应考虑这些参数中的每一个。