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

在基因治疗临床研究中，检测病毒/载体脱落是保证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样本中的复制型腺病毒（RCA）和腺病毒载体（AdV），我们试图建立人血液样本中复制型腺病毒（AdV）和复制型腺病毒载体（AdV）的检测方法。首先，建立感染性PCR检测RCA和AdV。该方法将RCA或AdV样本感染293细胞，通过实时荧光定量PCR对扩增的RCA或AdV进行定量。我们发现，感染性PCR可用于快速、灵敏地检测RCA和AdV。然而，感染性PCR在血液样本中检测RCA和AdV的效果并不好。因此，我们建立了利用pei磁珠和磁板对细胞进行高效感染的强制感染方法。我们还建立了另一种用G蛋白和抗腺病毒抗体强制感染的方法。采用PEI-beads法，AdV感染效率提高100倍以上。我们采用感染性PCR和PEI-beads感染相结合的方法检测血样中的RCA和AdV。然而，血液样本严重抑制AdV和RCA感染。因此，需要另一种方法更灵敏地检测血液样本中的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.