Development and validation of a model gene therapy biodistribution assay for AVGN7 using digital droplet polymerase chain reaction.
Development and validation of a model gene therapy biodistribution assay for AVGN7 using digital droplet polymerase chain reaction.
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DOI：
10.1016/j.omtm.2023.05.007
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发表时间：
2023-06-08
期刊：
影响因子：
--
通讯作者：
Rocha, Agostinho G.
中科院分区：
文献类型：
--
作者：
Rodgers, Buel D.;Herring, Sarah K.;Carias, Dereck R.;Chen, Joyce;Rocha, Agostinho G.
生物分布分析是基因治疗商业化不可或缺的一部分，传统上使用实时定量聚合酶链式反应。然而，液滴数字聚合酶链式反应(DdPCR)具有明显的优点，包括更高的灵敏度和绝对定量，但由于缺乏规范指导和文献中有意义的例子而未得到充分利用。我们报告了一个适用于目的的模型过程，以验证符合良好的实验室实践(GLP)的AVGN7的ddPCR检测，AVGN7是一种治疗肌肉萎缩的Smad7基因。利用gBlock DNA对Smad7和小鼠TATA盒结合蛋白的双重引物/探针集进行了优化，动态范围为10-80,000拷贝/反应在250 ng小鼠gDNA中。用线性化的质粒和小鼠gDNA进行验证，测定了精密度、准确度、粗糙度/稳健性、选择性、回收率、特异性、稀释线性和稳定性。批间精密度和准确度符合先前建立的标准，−偏差在5%~15%之间，变异系数(CV)小于19%，总误差在8%~35%之间。该方法的检测下限为2.5拷贝/反应，线性范围为40-80,000拷贝/反应，通过单个液滴群体证明了特异性，并证明了台式、冷藏和重复冻融循环的检测稳定性。提供的程序路线图超出了最近制定的标准。它还与许多支持IND的过程相关，因为经过验证的ddPCR分析可用于生物分布研究以及媒介滴定和制造质量控制。罗杰斯和他的同事描述了一种开发和验证水滴数字聚合酶链式反应(DdPCR)生物分布分析的方法，该方法用于量化不同组织中的基因治疗。这提供了在没有监管当局指导文件的情况下的程序路线图。
Biodistribution assays are integral to gene therapy commercialization and have traditionally used real-time qPCR. Droplet digital PCR (ddPCR), however, has distinct advantages including higher sensitivity and absolute quantification but is underused because of lacking regulatory guidance and meaningful examples in the literature. We report a fit-for-purpose model process to validate a good laboratory practice (GLP)-compliant ddPCR assay for AVGN7, a Smad7 gene therapeutic for muscle wasting. Duplexed primer/probe sets for Smad7 and mouse TATA-box binding protein were optimized using gBlock DNA over a dynamic range of 10–80,000 copies/reaction in 250 ng mouse gDNA. Linearized plasmid and mouse gDNA were used for validation, which determined precision, accuracy, ruggedness/robustness, selectivity, recovery, specificity, dilution linearity, and stability. Inter-run precision and accuracy met previously established criteria with bias between −5% and 15%, coefficient of variation (CV) less than 19%, and total error within 8%–35%. The limit of detection was 2.5 copies/reaction, linearity was confirmed at 40–80,000 copies/reaction, specificity was demonstrated by single droplet populations and assay stability was demonstrated for benchtop, refrigerated storage, and repeated freeze-thaw cycles. The procedural road map provided exceeds recently established standards. It is also relevant to many IND-enabling processes, as validated ddPCR assays can be used in biodistribution studies and with vector titering and manufacturing quality control. Rodgers and colleagues describe a method for developing and validating droplet digital PCR (ddPCR) biodistribution assays that are used to quantify gene therapeutics in different tissues. This provides a procedural road map in the absence of regulatory authority guidance documents.
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