Recombinant adeno‐associated virus (rAAV) is the leading platform of gene delivery for its long‐lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time‐consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single‐stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double‐stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual‐reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

中文翻译：

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用于理解和改进 AAV 基因组大小分析的双参考研究设计

重组腺相关病毒（rAAV）因其持久的基因转化和低免疫原性而成为领先的基因递送平台。 rAAV 基因组的完整性和纯度的表征对于确保临床效力和安全性至关重要。然而，目前可以提供大小评估的 rAAV 基因组表征方法要么耗时，要么不易被一般实验室使用。此外，缺乏正确的参考标准来分析长单链 DNA (ssDNA) 片段。在这里，我们使用 ssDNA 参考标准在微流体毛细管电泳平台上开发了 ssDNA 测定。该测定提供了 ssDNA 片段的大小调用，检测灵敏度为 ∼89 pg/μL (3 × 1010GC/mL AAV）用于 5.1 kb ssDNA 片段，每个样品的周转时间约为 100 秒，具有高通量样品分析能力。此外，我们观察到 AAV ssDNA 从衣壳释放后的退火可能会引入额外的双链 DNA (dsDNA) 峰。这种现象取决于样品处理流程。为了避免错误表征的风险，我们建议将双参考标准与其他正交方法结合使用，以全面了解 rAAV 基因组大小和完整性。