Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro.
BACKGROUND
Human adenoviruses (Ads) have substantial potential for clinical applications in cancer patients. Conditionally replicating adenoviruses (CRAds) include oncolytic adenoviruses in which expression of the immediate early viral transactivator protein E1A is controlled by a cancer cell-selective promoter. To enhance efficacy, CRAds are further armed to contain therapeutic genes. Due to size constraints of the capsid geometry, the capacity for packaging transgenes into Ads is, however, limited. To overcome this limitation, the employment of E1A-deleted replication-deficient viruses carrying therapeutic genes in combination with replication-competent CRAd vectors expressing E1A in trans has been proposed. Most trans-complementing studies involved transgene expressions from strong ubiquitous promoters, and thereby relied entirely on the cancer cell specificity of the CRAd vector.


RESULTS
Here we tested the trans-complementation of a CRAd and a replication-deficient transgene vector containing the same cancer cell-selective promoter. Hereto, we generated two new vectors expressing IL-2 and CD40L from a bicistronic expression cassette under the control of the melanoma/melanocyte-specific tyrosinase enhancer tyrosinase promoter (TETP), which we previously described for the melanoma-specific CRAd vector AdDeltaEP-TETP. These vectors gave rise to tightly controlled melanoma-specific transgene expression levels, which were only 5 to 40-fold lower than those from vectors controlled by the nonselective CMV promoter. Reporter analyses using Ad-CMV-eGFP in combination with AdDeltaEP-TETP revealed a high level of trans-complementation in melanoma cells (up to about 30-fold), but not in non-melanoma cells, unlike the AdCMV-eGFP/wtAd5 binary vector system, which was equally efficient in melanoma and non-melanoma cells. Similar findings were obtained when replacing the transgene vector AdCMV-eGFP with AdCMV-IL-2 or AdCMV-CD40L. However, the combination of the novel AdTETP-CD40L/IL-2 vector with AdDeltaEP-TETP or wtAd5 gave reproducible moderate 3-fold enhancements of IL-2 by trans-complementation only.


CONCLUSIONS
The cancer cell-selective TETP tested here did not give the expected enforceable transgene expression typically achieved in the Ad trans-complementing system. Reasons for this could include virus-mediated down regulation of limiting transcription factors, and/or competition for such factors by different promoters. Whether this finding is unique to the particular promoter system tested here, or also occurs with other promoters warrants further investigations.
Fontecedro
Alessandra Curioni
AC
Faculty of Mathematics and Natural Sciences, Institute of Molecular Life Sciences, University of Zürich, and Department of Dermatology, University Hospital of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Lutschg
Verena
V
Eichhoff
Ossia
O
Dummer
Reinhard
R
Greber
Urs F
UF
Hemmi
Silvio
S
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D000818:Animals; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D015316:Genetic Therapy / Q000295:instrumentation; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology / Q000235:genetics*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014779:Virus Replication
D007376:Interleukin-2; D023201:CD40 Ligand; D014442:Monophenol Monooxygenase