Development of sensitive detection methods for viral shedding of adenovirus vector and replication-competent adenovirus

病毒/载体脱落检测是基因治疗临床研究中腺病毒载体产品安全性的重要问题之一。为了更灵敏、快速地检测患者血液标本中的可复制型腺病毒（RCA）和腺病毒载体（AdV），本研究尝试建立人血液标本中AdV和RCA的检测方法，首先建立了RCA和AdV的感染性PCR检测方法，用RCA或AdV感染293细胞，用实时荧光PCR定量扩增RCA或AdV。结果表明，感染性PCR可用于RCA和AdV的快速、灵敏检测，但对血液标本中的RCA和AdV检测效果不佳。因此，我们建立了PEI-磁珠和磁性板的强制感染方法，以高效地感染细胞。我们还建立了另一种用G蛋白和抗腺病毒抗体进行强制感染的方法。将PEI-磁珠法与感染性PCR相结合，检测血液标本中的RCA和AdV。然而，AdV和RCA的感染被血液样品严重抑制。因此，需要另一种方法来更灵敏地检测血液样品中的RCA和AdV。

Detection of virus/vector shedding is one of the most important issues for safety of adenovirus vector products in gene therapy clinical research. In order to detect replication-competent adenovirus (RCA) and adenovirus vector (AdV) in blood samples of patients more sensitively and rapidly, we tried to establish a detection methods for AdV and RCA in human blood samples.First, infectivity PCR was established for the detection of RCA and AdV. In this method, 293 cells were infected with RCA or AdV samples, and amplified RCA or AdV were quantified by real-time PCR. We showed that infectivity PCR was useful for the rapid and sensitive detection of RCA and AdV. However, infectivity PCR did not work well to detect RCA and AdV in blood samples. Therefore, we then established forcibly infection method using PEI-magnetic beads and magnetic plate for highly effective infection to cells. We also establish another forcibly infection method using protein G and anti-adenovirus antibody. Using PEI-beads method, infection efficiency of AdV increased more than 100 times.We combined infectivity PCR and PEI-beads infection for detection of RCA and AdV in blood samples. However, infection or AdV and RCA was severely inhibited by blood samples. Therefore, another approach is required to detect RCA and AdV in blood samples more sensitively.