Department of Pediatrics, School of Medicine, Stanford University, Stanford, California, USA.
Department of Genetics, School of Medicine, Stanford University, Stanford, California, USA.
Hum Gene Ther. 2020 May;31(9-10):553-564. doi: 10.1089/hum.2019.339. Epub 2020 Mar 5.
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.
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，但仍存在一些限制，需要进一步改进。一些挑战包括存在不同水平的预先存在的中和抗体和在特定靶组织和/或疾病中的转导效率差。此外，由于针对衣壳的免疫反应，通常不可能再次施用 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 选择的过程尚不清楚，需要新的策略来确定开发更有效和功能性的 rAAV 衣壳的最佳参数。因此，使用条形码对 AAV 衣壳文库进行筛选，通过高通量测序进行筛选，为跟踪 AAV 衣壳进化并可能改进 AAV 衣壳文库筛选提供了强大的工具。在这项研究中，我们研究了不同参数如何影响两种不同 AAV 文库在两种人类细胞类型中的筛选。我们揭示了如何最大程度地提高获得具有所需特性的 AAV 变体的可能性的新的和意外的见解。该研究的主要发现如下。（1）包含 AAV 复制的辅助病毒可以选择性地繁殖能够复制到更高滴度的变体，但不一定更擅长转导。（2）具有特定衣壳的 AAV 之间的竞争可以在已经感染不同 AAV 的细胞中发生。（3）使用低感染复数进行感染会导致筛选之间的差异更大，并且不是选择最理想衣壳的最佳选择。（4）使用多轮选择可能适得其反。我们得出的结论是，在筛选具有增强的特性的 AAV 文库时，应该考虑到这些参数中的每一个。
