Shevade Aishwarya, Reeves John S, Bak Hanne, Tustian Andrew D
Preclinical Manufacturing and Process Development, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Mol Ther Methods Clin Dev. 2025 Jul 15;33(3):101529. doi: 10.1016/j.omtm.2025.101529. eCollection 2025 Sep 11.
Recombinant, non-replicative adeno-associated virus (AAV) containing a therapeutic gene of interest (GOI) is widely used as a vector for gene therapy. One way to manufacture AAV is through triple-transfection of HEK293 cells, with plasmids containing the (1) GOI, (2) replication ( and capsid () sequences, and (3) adenovirus helper sequences. During the manufacturing of AAV, replication-competent AAV (rcAAV) could theoretically be generated via homologous and non-homologous recombination events. rcAAV contaminants could lead to reduced efficacy, or an adverse immunogenic response. Therefore, testing is required by regulatory agencies. However, there is a paucity of literature on this critical assay. Here, we have developed a sensitive, cell-based assay for detection of rcAAV in AAV8 preparations. After transducing HEK293 cells over three rounds with AAV8 in the presence of helper adenovirus 5, we performed qPCR to detect the presence of rcAAV using the gene as a marker. The optimized assay is performed at a 2 mL scale, minimizes the generation of false-positive results, and achieves a reportable result of 1 rcAAV per 10 vector genomes. The same approach for rcAAV method development can be expanded to all other AAV serotypes, providing means for substantially improving process development and product safety.
含有目的治疗基因（GOI）的重组、非复制型腺相关病毒（AAV）被广泛用作基因治疗的载体。生产AAV的一种方法是通过对HEK293细胞进行三重转染，使用含有（1）GOI、（2）复制（和衣壳（）序列以及（3）腺病毒辅助序列的质粒。在AAV生产过程中，理论上可通过同源和非同源重组事件产生具有复制能力的AAV（rcAAV）。rcAAV污染物可能导致疗效降低或产生不良免疫原性反应。因此，监管机构要求进行检测。然而，关于这种关键检测方法的文献很少。在这里，我们开发了一种灵敏的基于细胞的检测方法，用于检测AAV8制剂中的rcAAV。在用辅助腺病毒5存在的情况下用AAV8对HEK293细胞进行三轮转导后，我们进行qPCR，以 基因作为标志物检测rcAAV的存在。优化后的检测方法以2 mL规模进行，最大限度地减少假阳性结果的产生，并实现每10个载体基因组中有1个rcAAV的可报告结果。rcAAV方法开发的相同方法可扩展到所有其他AAV血清型，为大幅改进工艺开发和产品安全性提供手段。
