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. 2010 Jul 29:7:175.
doi: 10.1186/1743-422X-7-175.

Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro

Alessandra Curioni Fontecedro  1 , Verena Lutschg, Ossia Eichhoff, Reinhard Dummer, Urs F Greber, Silvio Hemmi
Affiliations

Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro

Alessandra Curioni Fontecedro et al. Virol J. .

Abstract

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.

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Figures

Figure 1
Figure 1
Structures of E1A promoter of wt Ad5, AdΔEP-TETP and E1 region of AdCMV-eGFP, AdTETP-CD40L/IL-2. (A) The first 650 bp of the wt Ad genome comprising the left ITR, enhancer elements (EI, EII), packaging elements (I-VII), minor (thin arrows) and major (bold arrow) transcriptional start sites, TATA-box and the beginning of the E1A ORF. (B) Insertion of TETP in combination with duplication of packaging elements V-VII and deletion of endogenous E1A promoter resulting in AdΔEP-TETP. (C) In AdCMV-eGPF, the deleted E1 region from nt 449 to 3333 is replaced with the CMV-eGPF expression cassette. (D) In AdTETP, the deleted E1 region from nt 449 to 3333 is replaced with the TETP-CD40L-IRES-IL-2 expression cassette. The nucleotide numbers refer to the corresponding wt sequence.
Figure 2
Figure 2
CD40L transgene expression by different recombinant Ad vectors. Serial 3-fold increasing amounts of AdTETP-IL-2/CD40L (A), AdCD40L/IL-2 (B), and AdCMV-CD40L (C) were used to transduce HeLa cervical carcinoma, SW480 colon carcinoma, and M000301 and M21L4 melanoma cells for 5 h. Cells were then washed, and CD40L was analyzed two days p.i. by flow cytometry. Results are shown as mean fluorescence intensity (MFI) of triplicate measurements. (D) Transduction of M000301 cells with AdCD40L/IL-2 without washing off virus. Analysis was performed as described for A-C.
Figure 3
Figure 3
IL-2 transgene expression by different recombinant Ad vectors. Serial 3-fold increasing amounts of AdTETP-IL-2/CD40L (A), AdCD40L/IL-2 (B), and AdCMV-IL-2 (C) were used to transduce HeLa cervical carcinoma and M000301 melanoma cells for 5 h. Cells were then washed, and IL-2 of the supernates was analyzed two days p.i. by ELISA. Results are shown as mean of duplicate measurements.
Figure 4
Figure 4
Concentration-dependence and kinetics of Ad co-replication-mediated enhancement of eGFP expression. SW480 cells were infected with AdCMV-eGFP at MOIs of 0.37, 1.1, 3.3 and 10 alone (-), or in combination with wtAd5 at concentrations ranging from MOI 0.37 to 90, using serial 3-fold increases. The resulting ratios of wtAd5/AdCMV-eGFP are indicated. eGFP expression analyses were performed at day 1, 2, 3 and 4 p.i. by flow cytometry, and enhancement factors were calculated, following subtraction of auto-fluorescence of uninfected cells (cells only, co), unless, differences between uninfected and infected cells at MOI 0.37 were statistically not significant (not determined, nd).
Figure 5
Figure 5
Comparison of co-replication enhancement by wtAd5 and melanoma RC Ad virus AdΔEP-TETP. The indicated non-melanoma and melanoma cells were co-infected using AdCMV-eGFP at MOIs of 0.37, 1.1, 3.3 and 10 in combination with RC wtAd5 and AdΔEP-TETP, or the RD E1-deleted AdCMV-lacZ. Concentrations of the latter viruses were in the range from MOI 0.37 to 90, using serial 3-fold increases and resulting virus ratios as in Fig. 4. eGFP expression was recorded at day 2 by flow cytometry, and enhancement factors were calculated as in Fig. 4.
Figure 6
Figure 6
Adenovirus co-replication enhancement of immune modulators CD40L and IL-2 expressed from CMV promoter cassette. Melanoma M000301 or colon SW480 cells were infected with AdCMV-CD40L (A, B), or AdCMV-IL-2 (C), at an MOI of 1.1, and were combined with 3-fold increasing amounts of RC wtAd5 and AdΔEP-TETP, or the replication-defective E1-deleted AdCMV-lacZ. The resulting ratios of RC/transgene vector are indicated. CD40L and IL-2 transgene expression analyses were performed two days p.i. by flow cytometry and ELISA, respectively. Asterisks indicate the level of significance (*P < 0.05; **P < 0.005; for comparison with corresponding wtAd5/AdCMV-CD40L values).
Figure 7
Figure 7
Adenovirus co-replication enhancement of immune modulators CD40L and IL-2 expressed from the tissue-specific TETP promoter cassette. Melanoma M000301 cells were infected with AdTETP-CD40L/IL-2 at the indicated MOIs and were combined with 3-fold increasing amounts of RC AdΔEP-TETP (A, B), or wtAd5 (C, D). The resulting ratios of RC/transgene vector are indicated. CD40L and IL-2 transgene expression analyses were performed two days p.i.. This experiment was performed twice, with similar results.
Figure 8
Figure 8
Mitf RNA expression levels in non-infected (co) and infected cells as measured by RT-PCR. Cells were harvested two days p.i..
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