Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors
Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors
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DOI：
10.1089/hgtb.2019.031
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发表时间：
2019-06-21
影响因子：
--
通讯作者：
Uchida, Eriko
中科院分区：
文献类型：
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作者：
Furuta-Hanawa, Birei;Yamaguchi, Teruhide;Uchida, Eriko
重组腺相关病毒（rAAV）载体最近已被广泛用于体内基因治疗。AAV载体的临床剂量定义需要作为起始剂量和用于剂量递增研究的精确定量。通过定量PCR（qPCR）测量的载体基因组（vg）拷贝通常用于rAAV载体滴定，并且rAAV载体质粒DNA通常用于qPCR标准品，尽管血清型2和8的rAAV参考标准物质（RSM）（rAAV 2 RSM和rAAV 8 RSM）可从美国典型培养物保藏中心获得。然而，基于qPCR的AAV vg测定受qPCR标准品和扩增靶位点选择的影响。在这项研究中，我们已经开发了一种新的PCR方法，二维液滴数字PCR（2D ddPCR），用于目标DNA的绝对定量和用于评估rAAV载体的稳定性。当用限制性内切酶处理具有不同构象的标准质粒DNA时，通过qPCR测定的rAAV 2 RSM的vg拷贝数显著改变，表明qPCR扩增显著受标准品二级结构的影响。相比之下，通过ddPCR确定的vg拷贝数不受针对靶位点的不同位置使用引物探针或vg的二级结构构象的影响。此外，AAV vg的完整性可以使用2D ddPCR用荧光素和六氯-6-羧基荧光素标记的探针靶向相同rAAV基因组中的不同位置来监测。在加速（37 ℃）稳定性研究中，完整rAAV的滴度与rAAV活性高度相关。2D ddPCR是rAAV载体定量和质量评价的有用工具。
Recombinant adeno-associated virus (rAAV) vectors have recently been widely utilized for in in vivo gene therapy. The clinical dose definition of AAV vector requires the exact quantification as starting doses and for dose-escalation studies. Vector genome (vg) copies measured by quantitative PCR (qPCR) are commonly used for rAAV vector titration, and rAAV vector plasmids DNA is often used for qPCR standards, although the rAAV reference standard materials (RSMs) for serotypes 2 and 8 (rAAV2RSM and rAAV8RSM) are available from American Type Culture Collection. However, qPCR-based determination of the AAV vg is affected by the selection of the qPCR standard and the amplification target sites. In this study, we have developed a new PCR method, two-dimensional droplet digital PCR (2D ddPCR), for the absolute quantitation of target DNA and for evaluating the stability of the rAAV vector. The number of vg copies of rAAV2RSM determined by qPCR dramatically changed when standard plasmid DNAs with different conformations were treated with restriction enzymes, suggesting that qPCR amplification is significantly affected by the secondary structure of the standard. In contrast, the number of vg copies determined by ddPCR was unaffected by using primer probes for different positions of target sites or by the secondary structure conformation of the vg. Furthermore, the integrity of the AAV vg can be monitored using 2D ddPCR with fluorescein- and hexachloro-6-carboxy-fluorescine-labeled probes targeting different positions in the same rAAV genome. The titer of intact rAAV was highly correlated with rAAV activity in an accelerated (37 degrees C) stability study. 2D ddPCR is a useful tool for rAAV vector quantitation and quality evaluation.