Linked Tumor-Selective Virus Replication and Transgene Expression from E3-Containing Oncolytic Adenoviruses

Abstract

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/⌬E3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/⌬E3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression.

Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression

“…Strategies that utilize the adenoviral transcriptional machinery by either replacing adenoviral coding regions of specific genes 15,18,22,32 or linking transgene expression via consensus splice acceptors sites 21 have recently been reported. The latter approach has two main benefits: First, by using the endogenous adenoviral sequences to control transgene expression, as opposed to utilizing exogenous promoters, the overall size of the transgene cassette can be reduced, reducing the constraints on effective packaging of the adenoviral genome.…”

“…38 Second, by linking transgene expression to the endogenous adenoviral transcriptional elements, transgene expression will closely mimic that of the adenoviral genes in both temporal expression patterns and, importantly, expression levels. 15,[18][19][20]22,32 Furthermore, if transgenes are inserted into adenoviral regions that are expressed after DNA replication, transgene expression can be restricted to sites of viral replication, which, in the case of tumor-specific oncolytic viruses, means that expression is restricted to tumors. This provides the opportunity to arm oncolytic viruses with cytotoxic transgenes, but to maintain the vectors' safety profile.…”

“…The E3 region was chosen as an insertion site in two vectors on the basis of previous studies demonstrating that E3 genes are not essential for viral replication and that transgenes can be successfully inserted into this region, without major impairment of viral function. 18,20,22,32 In the Ad-GM-gp19K vector, the hGM-CSF cDNA was inserted into the E3 gp19K region as previously described for the vector Dgp19B. 22 The hGM-CSF cDNA was inserted in place of the E3 14.7K gene in the second E3-based vector, Ad-GM-14.7.…”

“…18,20,22,32 In the Ad-GM-gp19K vector, the hGM-CSF cDNA was inserted into the E3 gp19K region as previously described for the vector Dgp19B. 22 The hGM-CSF cDNA was inserted in place of the E3 14.7K gene in the second E3-based vector, Ad-GM-14.7. Given the known overlap in coding sequences between the E3 14.7K gene and the upstream E3 14.5K gene in the wild-type adenovirus 33 genome the hGM-CSF cDNA was inserted in the E3 14.7K region in a manner to both optimize GM-CSF expression and maintain the integrity of the E3 14.5K/14.7K overlap sequence.…”

“…15,18 This tumor-specific transgene expression improves the safety of the vector. Several insertion sites, such as the E3A region 11,18,22 and E3B region, 15,18 were identified on the basis of detailed knowledge of transcription in adenoviruses. More recently, a novel transposonbased mechanism has been described that identified other potential insertion sites, even though the exact transcriptional regions had not been previously identified.…”