Takino Rie, Yamaguchi Yuki, Maruno Takahiro, Ramadhani Ekaputra, Furukawa Misaki, Torisu Tetsuo, Uchiyama Susumu
Analytical and Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Mol Ther Methods Clin Dev. 2024 Oct 28;32(4):101362. doi: 10.1016/j.omtm.2024.101362. eCollection 2024 Dec 12.
Recombinant adeno-associated virus (rAAV) is a small, nonenveloped virus vector that has attracted attention as a gene therapy platform. Several studies have revealed the stability characteristics of rAAV; however, its tolerance against light stress remains unclear. Here, the physicochemical and biological impacts of light stress on rAAV6 with EGFP as a gene of interest were investigated. The results revealed that light exposure led to an approximately 20% loss in particle number of rAAV with value of full particles, whereas a more drastic decrease was observed in the genomic titer due to the degradation of encapsidated DNA, leading to an approximately 90% reduction in biological activity as evident from a flow cytometry-based assay. Structure analysis of the viral proteins VP1, VP2, and VP3 and the encapsidated DNA revealed that light exposure causes typical photodegradation of protein and DNA, but with AAV-specific phenomena. This included oxidation proceeding at histidine residues of VP3 on the inner surface of the capsid and the formation of cyclobutane pyrimidine dimer in DNA within the capsid. The findings highlight the necessity of the proper handling and storage to maintain the stability and efficacy of AAV-based gene therapies.
重组腺相关病毒（rAAV）是一种小型无包膜病毒载体，作为一种基因治疗平台已引起关注。多项研究揭示了rAAV的稳定性特征；然而，其对光应激的耐受性仍不清楚。在此，研究了光应激对以EGFP为目的基因的rAAV6的物理化学和生物学影响。结果显示，光照导致rAAV全颗粒数量损失约20%，而由于衣壳内包裹的DNA降解，基因组滴度出现更显著下降，基于流式细胞术的检测表明其生物活性降低了约90%。对病毒蛋白VP1、VP2和VP3以及衣壳内包裹的DNA进行结构分析发现，光照会导致蛋白质和DNA发生典型的光降解，但存在AAV特异性现象。这包括衣壳内表面VP3的组氨酸残基发生氧化过程，以及衣壳内DNA中形成环丁烷嘧啶二聚体。这些发现凸显了正确处理和储存以维持基于AAV的基因治疗的稳定性和疗效的必要性。
