Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)—AAVS1, CCR5, and human ROSA26 loci—were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with recombinase-mediated cassette exchange.

中文翻译：

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综合分析基因组安全港作为HEK293细胞中稳定表达异源基因的目标位点。

人类细胞系由于其人类糖基化机制而越来越多地被用作产生治疗性糖蛋白的宿主细胞。为了开发一种平台，用于生成基于靶向整合的可产生治疗性蛋白的等基因人类细胞系，针对转基因表达，评估了三个著名的人类基因组安全港（AASH1，CCR5和人类ROSA26基因座）人胚胎肾（HEK293）细胞中的水平和稳定性。在三个GSH中，AAVS1基因座显示出最高的eGFP表达和最高的同质性。AAVS1基因座的转基因表达持续进行了约3个月，没有选择。此外，CMV启动子显示最高的表达，其后是AAVS1基因座的EF1α，SV40和TK启动子。