Luo Yuxia, Frederick Amy, Martin John M, Scaria Abraham, Cheng Seng H, Armentano Donna, Wadsworth Samuel C, Vincent Karen A
1 Sanofi Genzyme , Framingham, Massachusetts.
Hum Gene Ther Methods. 2017 Jun;28(3):124-138. doi: 10.1089/hgtb.2016.158. Epub 2017 Apr 14.
Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.
腺相关病毒（AAV）生产细胞系是通过用包含三个组件（载体序列、AAV rep和cap基因以及一个选择标记基因）的单个质粒转染HeLaS3细胞而创建的。由于该质粒同时包含位点特异性整合所需的顺式（Rep结合位点）和反式（由rep基因编码的Rep蛋白）元件，因此预计质粒整合可能发生在人类19号染色体（chr19）上的AAVS1位点内。本研究的目的是调查AAVS1中的整合是否可能与载体产量相关。通过Southern印迹、荧光原位杂交（FISH）和PCR分析评估了几个独立细胞系中的质粒整合位点。在Southern印迹分析中，rep特异性探针和AAVS1特异性探针均检测到片段的存在，这表明对于几个中高产细胞系，质粒DNA已整合到AAVS1位点。用嘌呤霉素抗性基因（puroR）特异性探针和AAVS1特异性探针进行的分析表明，AAVS1中的整合是一种更普遍的现象。使用针对质粒、AAVS1和chr19标记的特异性探针，通过FISH对高产量的AAV2分泌碱性磷酸酶（SEAP）细胞系（主孔82 [MW82]和MW278）进行了评估。FISH分析在MW278中检测到两个质粒整合位点（均不在AAVS1中），而在MW82中总共鉴定出三个位点（两个在AAVS1中）。反向PCR分析证实了几个中高产细胞系在AAVS1内的整合。总之，FISH、Southern印迹和PCR数据提供了证据，表明质粒在几个AAV生产细胞系的AAVS1内发生了位点特异性整合。数据还表明，AAVS1中的整合是一种普遍现象，不一定仅限于高产细胞系。结果还表明，AAVS1位点内的质粒整合不是高载体产量的绝对必要条件。
