Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials.</p> <p>Results</p> <p>The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The <it>in vivo </it>assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the <it>in vitro </it>studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.</p> <p>Conclusions</p> <p>These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.</p

Attenuation of Vaccinia Tian Tan Strain by Removal of Viral TC7L-TK2L and TA35R Genes

Vaccinia Tian Tan (VTT) was attenuated by deletion of the TC7L-TK2L and TA35R genes to generate MVTT3. The mutant was generated by replacing the open reading frames by a gene encoding enhanced green fluorescent protein (EGFP) flanked by loxP sites. Viruses expressing EGFP were then screened for and purified by serial plaque formation. In a second step the marker EGFP gene was removed by transfecting cells with a plasmid encoding cre recombinase and selecting for viruses that had lost the EGFP phenotype. The MVTT3 mutant was shown to be avirulent and immunogenic. These results support the conclusion that TC7L-TK2L and TA35R deletion mutants can be used as safe viral vectors or as platform for vaccines

Immune responses induced by vaccination.

<p>Ten 6-week-old BALB/c mice received 5×10⁴ PFU MVTT1, MVTT2 or MVTT3 by intramuscular route and three weeks later received booster of the same dose in 0.1 ml of PBS. The serum were harvested on weeks 3 and 5 post infection (i.m.). Shown were induced IL-2, IL-4, IL-10 and IFN-γsystemic immune responses in the two vaccinated cohorts as measured by mouse ELISA kit analysis. All measurements were performed in triplicate. Data were presented as mean ± SD.</p

Virus titer of mutants in five cell lines tested.

<p>PK(15), MDCK, HeLa, BHK-21 and Vero cells infected with 0.05 MOI of VTT and the mutants, and then the viruses were harvested and titrated in BHK-21 cells. Virus titer was determined by measuring the plaque assays.</p

Schematic representation of the MVTT3 genome and identification of mutants.

<p>(A) The TC7L-TK2L and TA35R genomic deletions were found in the viral genome. (B) EGFP was expressed by mutants MVTT1-EGFP, MVTT2-EGFP, or MVTT3-EGFP in the BHK-21 cells. The virus-infected cells were visualized by isolated fluorescent plaque, which was recognized in the same visual fields. Non-fluorescent plaques appeared because of recombinant MVTT1, MVTT2, or MVTT3 in the BHK-21 cells. All photos were taken at 200× magnification.</p

PCR analysis results of the TC7L-TK2L and TA35R genes of the isolated mutant.

<p>Diagnostic PCR was performed to identify the final mutant. The deletion of the TC7L-TK2L and TA35R genes was investigated by PCR 72 h after infection of BHK-21 cells with 0.01 MOI of wild-type VTT. Approximately 366 bp (TC7L-TK2L; A, lane 1) and 353 bp (TA35R; A, lane 3) were detected by ethidium bromide staining. In addition, the TC7L-TK2L and TA35R fragments were missing in the mutants MVTT1-EGFP (TC7L-TK2L; A, lane 2), MVTT2-EGFP (TA35R; A, lane 4), and MVTT3-EGFP (TC7L-TK2L and TA35R; A, lane 5 and lane 6). The genes 431 bp flanking the TC7L-TK2L regions (B, MVTT1, lane 1; and MVTT3, lane 3) and the genes 1142 bp flanking the TA35R regions (B, MVTT2, lane 2; and MVTT3, lane 4) were investigated to identify non-EGFP-expressing virus. Evaluation of the genetic stability in the BHK-21 cells after 2, 4, 6, 8 and 10 passages. TC7L-TK2L gene of MVTT1 (C, lanes 2–6), TA35R gene of MVTT2 (D, lane 2–6), TC7L-TK2L gene of MVTT3 (G, lanes 3–7), and TA35R gene of MVTT3 (G, lanes 8–12) produced negative results, comparing to positive PCR results. The genes of MVTT1 flanking the TC7L-TK2L regions (E), the genes of MVTT2 flanking the TA35R regions (F), and the genes of MVTT3 flanking the TC7L-TK2L regions (H, lanes 1–5) or TA35R regions (H, lanes 6–10) were detected correctly.</p

Plaque phenotypes formed by infection with mutant viruses.

<p>Confluent monolayers of PK(15), MDCK, HeLa, BHK-21 and Vero cells in 12-well plates were infected with 0.05 PFU/cell of MVTT1, MVTT2, MVTT3, or VTT viruses. The plates were incubated at 37°C for 2 d prior to staining with 0.1% crystal violet. The pathogenicity of MVTT1, MVTT2, and MVTT3 apparently decreased in all five cell lines (compared with VTT as the control).</p