测定重组腺相关病毒基因组滴度的qPCR新方法

Novel qPCR strategy for quantification of recombinant adeno-associated virus serotype 2 vector genome-titer

摘要:

腺相关病毒 (Adeno-associated virus，AAV) 在基因治疗应用中具有很多优势，但是其生物学滴度的测定仍很繁琐，不同实验室使用各自的方法和参照，这些都影响了重组腺相关病毒 (rAAV) 载体在临床前和临床上的应用。反向末端重复序列 (Inverted terminal repeats，ITR) 是重组腺相关病毒载体中不可或缺的顺式作用元件，针对ITR2以及ITR2-CMV设计的qPCR检测方法可以快速、准确地得到rAAV2的基因组滴度，由于该方法可以广泛适用，因此对推动AAV滴度检测的标准化有重要意义。

Abstract:

Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs’ vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.