Afione S A, Conrad C K, Kearns W G, Chunduru S, Adams R, Reynolds T C, Guggino W B, Cutting G R, Carter B J, Flotte T R
Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
J Virol. 1996 May;70(5):3235-41. doi: 10.1128/JVI.70.5.3235-3241.1996.
Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue.
基于人类DNA病毒的基因治疗载体可能会从随后感染相应野生型（wt）辅助病毒的个体中被激活或拯救出来。这种现象已在体外分别用腺病毒（Ad）和腺相关病毒（AAV）载体进行了有效模拟，但此前尚未在体内进行研究。在本研究中，我们建立了一个体内模型，以研究重组AAV载体（AAV-CFTR）与野生型2型AAV（AAV2）以及一种宿主范围突变型Ad（Ad2HR405，猴细胞对其敏感）（D.E.布劳、S.A.赖斯、S.塞尔和D.F.克莱西格，《病毒学杂志》55:206 - 212，1985年）之间的相互作用。将AAV-CFTR施用于恒河猴鼻腔或肺部的呼吸道上皮。在载体施用后长达3个月的时间点，从输注部位采集原代细胞，以确认载体DNA的持久性。载体DNA以游离形式存在，并且只有通过添加野生型AAV2和Ad才能在体外被拯救出来。在体内拯救研究中，在野生型AAV2和Ad2HR405感染之前或之后施用载体，并检测AAV-CFTR的释放情况。同时施用时，Ad2HR405和野生型AAV2在鼻腔中建立感染。当病毒以高滴度直接施用于下呼吸道时，野生型AAV2在肺部发生复制。在后一种情况下也观察到了AAV-CFTR载体的拯救。尽管每组动物数量较少，但似乎AAV-CFTR DNA在灵长类动物呼吸道中持续存在，并且该模型可能有助于重组AAV载体拯救的研究。
