Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

中文翻译：

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基于物理和功能转导的腺相关病毒载体的体外、体外和体内高通量筛选。

腺相关病毒 (AAV) 载体正迅速成为治疗基因传递的首选载体。迄今为止，已报道了数百种天然分离株和生物工程变体。虽然高生产滴度和低免疫反应性等因素很重要，但在为临床应用选择 AAV 变体时，传递基因有效载荷（物理转导）和驱动高转基因表达（功能转导）的能力仍然是最重要的特征。报告基因表达分析是确定载体适合度的最常用方法。然而，在评估大量变体时，此类方法既耗时又不切实际。对原始人体组织的有限访问或具有挑战性的模型系统进一步使矢量测试复杂化。为了解决这个问题，正在开发基于下一代测序 (NGS) 的便捷高通量方法。为此，我们构建了一个 AAV 测试套件，该套件在包含的 AAV 变体的数量和类型方面具有固有的灵活性，并且与体外、离体和体内应用。此处介绍的测试套件由 30 个已知 AAV 组成，其中每个变体编码一个 CMV-eGFP 盒和 eGFP 基因的 3' 非翻译区中的独特条形码，从而允许在 DNA 和 RNA/ cDNA 水平。为了验证 AAV 测试套件，将单独包装的条形码变体以相等的比例混合并用于转导感兴趣的细胞/组织。提取 DNA 和 RNA/cDNA，随后通过 NGS 进行分析以确定物理/功能转导效率。我们能够在体外和体内评估永生化细胞、原代细胞和诱导多能干细胞的转导效率在幼稚小鼠和异种移植肝脏模型中进行转导。重要的是，虽然我们的数据验证了先前报告的单个衣壳的转导特征，但我们还确定了一些 AAV 变体的新的先前未知的趋向性。