Le Long P, Li Jing, Ternovoi Vladimir V, Siegal Gene P, Curiel David T
Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, 901 19th Street South, BMR2-502, Birmingham, AL 35294, USA.
Departments of Pathology, Cell Biology and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-2172, USA.
J Gen Virol. 2005 Dec;86(Pt 12):3201-3208. doi: 10.1099/vir.0.80968-0.
Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX-enhanced green fluorescent protein (pIX-EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX-EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.
犬2型腺病毒（CAV2）因其无致病性且人群中不存在针对该病毒的预先存在的中和抗体，已成为基因治疗中一种有吸引力的载体。此外，该载体已被提议作为一种在骨钙素启动子控制下的条件性复制腺病毒制剂，用于在患有自发性骨肉瘤的同基因、免疫活性犬模型中进行评估。在本研究中，开发了一种用荧光衣壳融合蛋白IX-增强型绿色荧光蛋白（pIX-EGFP）标记的CAV2载体。可以检测到荧光融合蛋白标签在具有适当核定位的感染细胞中的表达，并整合到病毒粒子中。标记的病毒粒子可以通过荧光显微镜观察到；这适用于追踪CAV2感染，以及定位载体在组织中的分布。pIX-EGFP的表达可用于检测CAV2的复制和传播。这些结果表明，pIX可作为在犬腺病毒异型背景下整合异源蛋白的平台。据信，衣壳标记的CAV2在载体开发研究和监测基于CAV2的溶瘤腺病毒复制方面具有实用性。
