Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site

Jan E. Carette, Harm C A Graat, Frederik H E Schagen, Mohamed A I Abou El Hassan, Winald R. Gerritsen, Victor W. van Beusechem

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1053-1062
Number of pages10
JournalJournal of Gene Medicine
Volume7
Issue number8
DOIs
StatePublished - Aug 2005
Externally publishedYes

Fingerprint

RNA Splice Sites
Alternative Splicing
Transgenes
Adenoviridae
Luciferases
Cytomegalovirus
Oncolytic Viruses
Apigenin
Neoplasms
Flavonoids
Antineoplastic Agents
Genes
Genome
Viruses
Gene Expression
Polymerase Chain Reaction
Infection

Keywords

  • Alternative splicing
  • Conditionally replicating adenovirus
  • Gene therapy
  • Late transgene expression
  • Viral vector

ASJC Scopus subject areas

  • Genetics

Cite this

Carette, J. E., Graat, H. C. A., Schagen, F. H. E., Abou El Hassan, M. A. I., Gerritsen, W. R., & van Beusechem, V. W. (2005). Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site. Journal of Gene Medicine, 7(8), 1053-1062. https://doi.org/10.1002/jgm.754

Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site. / Carette, Jan E.; Graat, Harm C A; Schagen, Frederik H E; Abou El Hassan, Mohamed A I; Gerritsen, Winald R.; van Beusechem, Victor W.

In: Journal of Gene Medicine, Vol. 7, No. 8, 08.2005, p. 1053-1062.

Research output: Contribution to journalArticle

Carette, Jan E. ; Graat, Harm C A ; Schagen, Frederik H E ; Abou El Hassan, Mohamed A I ; Gerritsen, Winald R. ; van Beusechem, Victor W. / Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site. In: Journal of Gene Medicine. 2005 ; Vol. 7, No. 8. pp. 1053-1062.
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