Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.
To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.
Ono
Hidetaka A
HA
Division of Human Gene Therapy, Department of Medicine, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Le
Long P
LP
Davydova
Julia G
JG
Gavrikova
Tatyana
T
Yamamoto
Masato
M
D000230:Adenocarcinoma / Q000821:virology; D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D017503:Adenovirus E3 Proteins / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004261:DNA Replication; D005453:Fluorescence; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000821:virology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
D017503:Adenovirus E3 Proteins; C099609:adenovirus death protein, Adenovirus; D049452:Green Fluorescent Proteins