Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.
BACKGROUND
Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated.


METHODS
We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r).


RESULTS
The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations.


CONCLUSIONS
Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.
Le
Long P
LP
Division of Human Gene Therapy, Department of Medicine, University of Alabama, Birmingham, AL, USA.
Le
Helen N
HN
Dmitriev
Igor P
IP
Davydova
Julia G
JG
Gavrikova
Tatyana
T
Yamamoto
Seiji
S
Curiel
David T
DT
Yamamoto
Masato
M
D000230:Adenocarcinoma / Q000628:therapy*; D000260:Adenoviruses, Human / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D000818:Animals; D002213:Capsid; D036022:Capsid Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D042003:DNA Packaging; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008175:Lung Neoplasms / Q000628:therapy*; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000378:metabolism; D014183:Transplantation, Heterologous; D014779:Virus Replication; D000097573:Red Fluorescent Protein
D036022:Capsid Proteins; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D004279:DNA, Viral; D008164:Luminescent Proteins; D011991:Receptors, Virus; D000097573:Red Fluorescent Protein; C568771:CLMP protein, human; C568772:CLMP protein, mouse; C018315:hexon capsid protein, Adenovirus