Yu Lei, Zhou Yong, Shi Xin-Chang, Wang Guang-Yu, Fu Zhi-Hao, Liang Cheng-Gang, Wang Jun-Zhi
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, Liaoning 110016, P.R. China.
State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China.
Mol Ther Methods Clin Dev. 2024 Jul 22;32(3):101304. doi: 10.1016/j.omtm.2024.101304. eCollection 2024 Sep 12.
The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying the rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 10 and 10 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results ( < 0.05, t test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.
病毒基因组滴度是重组腺相关病毒（rAAV）基因治疗产品临床给药、生产和分析检测的关键指标。尽管定量PCR和数字PCR是用于定量rAAV基因组滴度的常用方法，但它们受到准确性和稳健性不足的限制。成簇规律间隔短回文重复序列（CRISPR）-Cas12a生物传感器在病毒检测中的应用越来越广泛；然而，目前尚无其在基因治疗产品滴度测定中应用的报道。在本研究中，开发、优化了一种无需扩增的CRISPR-Cas12a检测方法，并将其应用于rAAV基因组滴度的测定。该检测方法在4×10和10 vg/mL的检测范围内显示出高精度和准确性。Cas12a检测结果与qPCR检测结果之间未观察到显著差异（t检验，P<0.05）。此外，Cas12a在单链和双链DNA底物上均表现出相似的活性。基于这一特性，分别测定了正义链和反义链的滴度，结果显示，对于一种内部参考AAV5-IN，其正义链和反义链的滴度存在显著差异。本研究首次报道了一种用于rAAV基因组滴度测定和组成分析的Cas12a检测方法。
