Wang Yu, Menon Namrata, Shen Shen, Feschenko Marina, Bergelson Svetlana
Department of Analytical Development, Biogen, Inc., 225 Binney Street, Cambridge, MA 02142, USA.
Gene Therapy Accelerator Unit, Biogen, Inc., 225 Binney Street, Cambridge, MA 02142, USA.
Mol Ther Methods Clin Dev. 2020 Oct 1;19:341-346. doi: 10.1016/j.omtm.2020.09.017. eCollection 2020 Dec 11.
Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a "digestion-free" method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.
重组腺相关病毒（rAAV）是基因治疗中使用的主要载体之一。准确的基因组滴度不仅对临床给药至关重要，也是许多AAV产品特性分析检测的前提条件。传统上，AAV基因组滴度通过qPCR测定；然而，尽管付出了巨大努力，检测精度仍不理想。最近，液滴数字PCR（ddPCR）作为一种强大的替代方法出现，具有出色的准确性和精密度。然而，目前ddPCR不如qPCR普及，通量较低且成本较高。在本文中，我们介绍了一种改进的qPCR方法，该方法有两个主要优化：（1）使用AAV参考物质作为qPCR标准品，而不是质粒DNA；（2）通过在标准品和样品制备中添加5%吐温20实施“免消化”方法。新方法已针对不同血清型、纯化状态和携带转基因的AAV进行了广泛测试，结果发现该方法具有高度准确性、精密度和稳健性。这种显著改进和简化的检测方法可被基因治疗领域的研究人员轻松采用，并进一步实现高通量应用的自动化。
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