Böcker Wolfgang, Rossmann Oliver, Docheva Denitsa, Malterer Georg, Mutschler Wolf, Schieker Matthias
Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Munich, Germany.
J Gene Med. 2007 Jul;9(7):585-95. doi: 10.1002/jgm.1049.
Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs.
Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes.
Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages.
Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.
人间充质干细胞（hMSCs）是体外基因治疗的一个有前景的靶点，慢病毒是hMSCs中优秀的基因传递载体，因为它们能实现高转导率并具有长期基因表达。然而，hMSCs的衰老可能会限制治疗应用，因为细胞选择和病毒滴度测定耗时。在此，我们描述了一种快速可靠的方法，通过在hMSCs中高效体外基因转移后进行定量聚合酶链反应（qPCR）来测定功能性慢病毒滴度。
用不同类型的包装系统测试慢病毒生产。使用p24 ELISA在细胞培养上清液中检测剩余病毒颗粒。通过流式细胞术分析定量慢病毒基因转移效率。通过对表达的转基因进行qPCR来测定慢病毒滴度。
第三代自失活载体在hMSCs中显示出高效的基因转移。在更换四次培养基后，细胞培养上清液中未检测到病毒抗原，表明4天后不存在感染性颗粒。通过qPCR分析，我们观察到病毒稀释与转基因表达水平之间存在线性相关性，因此可以通过定量转基因表达来进行病毒滴度测定。最后，我们证明转导的hMSCs通过向脂肪生成、成骨和软骨生成谱系分化保留了其干细胞特性。
通过qPCR定量转基因拷贝数是一种快速可靠的方法，用于测定hMSCs体外基因转移后功能性慢病毒滴度。
