Adeno-associated viruses (AAVs) are advantageous as gene-transfer vectors due to their favorable biological and safety characteristics, with discovering novel AAV variants being key to improving this treatment platform. To date, researchers have isolated over 200 AAVs from natural sources using PCR-based methods. We compared two modern DNA polymerases and their utility for isolating and amplifying the AAV genome. Compared to the HotStar polymerase, the higher-fidelity Q5 Hot Start High-Fidelity DNA Polymerase provided more precise and accurate amplification of the input AAV sequences. The lower-fidelity HotStar DNA polymerase introduced mutations during the isolation and amplification processes, thus generating multiple mutant capsids with variable bioactivity compared to the input AAV gene. The Q5 polymerase enabled the successful discovery of novel AAV capsid sequences from human and nonhuman primate tissue sources. Novel AAV sequences from these sources showed evidence of positive evolutionary selection. This study highlights the importance of using the highest fidelity DNA polymerases available to accurately isolate and characterize AAV genomes from natural sources to ultimately develop more effective gene therapy vectors.

中文翻译：

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用高保真聚合酶从灵长类动物组织中分离天然腺相关病毒

腺相关病毒 (AAV) 作为基因转移载体是有利的，因为它们具有良好的生物学和安全特性，发现新的 AAV 变体是改进该治疗平台的关键。迄今为止，研究人员已使用基于 PCR 的方法从天然来源中分离出 200 多种 AAV。我们比较了两种现代 DNA 聚合酶及其用于分离和扩增 AAV 基因组的效用。与 HotStar 聚合酶相比，更高保真度的 Q5 热启动高保真 DNA 聚合酶对输入的 AAV 序列提供了更精确和准确的扩增。较低保真度的 HotStar DNA 聚合酶在分离和扩增过程中引入了突变，因此与输入的 AAV 基因相比，产生了具有可变生物活性的多个突变衣壳。Q5 聚合酶能够成功发现来自人类和非人类灵长类动物组织来源的新型 AAV 衣壳序列。来自这些来源的新型 AAV 序列显示出积极进化选择的证据。这项研究强调了使用最高保真度 DNA 聚合酶从天然来源中准确分离和表征 AAV 基因组以最终开发更有效的基因治疗载体的重要性。