Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site.
BACKGROUND
Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication.


METHODS
We constructed an oncolytic adenovirus that expresses luciferase under regulation of the endogenous major late promoter (MLP) via alternative splicing to an inserted splice-acceptor site analogous to that of the adenovirus serotype 40 long fiber gene. Splicing of the luciferase transcript was studied by RT-PCR analysis. Expression was measured in the presence and absence of the flavonoid apigenin, an inhibitor of viral replication.


RESULTS
The inserted splice-acceptor site was properly recognized by the adenoviral splicing machinery. Luciferase expression levels were markedly higher than levels obtained with the cytomegalovirus (CMV) promoter, especially at late stages of infection. Inhibiting adenovirus replication reduced luciferase expression levels dramatically by 4 to 5 logs, whereas expression levels with the CMV-luciferase adenovirus were only moderately affected (2 logs).


CONCLUSIONS
Transgene delivery using the endogenous late gene expression machinery resulted in an expression pattern distinct from expression driven by the conventional CMV promoter. The high expression levels and strict coupling of expression to viral replication should be useful for adequate monitoring of replication and might provide a platform for the design of armed conditionally replicating adenoviruses (CRAds) with enhanced oncolytic potency.
Carette
Jan E
JE
Division of Gene Therapy, Department of Medical Oncology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands. j.carette@vumc.nl
Graat
Harm C A
HC
Schagen
Frederik H E
FH
Abou El Hassan
Mohamed A I
MA
Gerritsen
Winald R
WR
van Beusechem
Victor W
VW
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology*; D017398:Alternative Splicing / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D050504:Oncolytic Viruses / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D022821:RNA Splice Sites / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D014779:Virus Replication / Q000502:physiology*
D022821:RNA Splice Sites