Potentiation of oncolytic adenoviral vector efficacy with gutless vectors encoding GMCSF or TRAIL.

Oncolytic adenoviral vectors selectively replicate in and lyse human tumor cells, providing a promising means for targeted tumor destruction. However, oncolytic vectors have limited capacity for incorporation of additional genetic material that could encode therapeutic transgenes and/or transcriptional regulatory control elements to augment the efficacy and/or safety of the vector. Therefore, we hypothesized that coadministration of an oncolytic vector with a replication-defective, gutless adenoviral vector encoding a therapeutic transgene would result in replication of both vectors within a tumor and potentiate antitumor efficacy relative to the use of either vector alone. We constructed gutless vectors encoding the murine granulocyte-macrophage colony-stimulating factor (AGVmGMF) or human tumor necrosis factor alpha-related apoptosis-inducing ligand (AGVhTRAIL) gene and tested the ability of these vectors to augment the efficacy of an oncolytic vector (Ar6pAE2fE3F) in a potentiating vector strategy. In Hep3B cells in vitro, cotreatment with Ar6pAE2fE3F increased transgene expression from AGVhTRAIL and permitted replication of AGVhTRAIL, suggesting that an oncolytic vector can propagate gutless vector spread in vivo. In pre-established Hep3B xenograft tumors, neither gutless vector alone inhibited tumor growth; however, coadministration of AGVmGMF or AGVhTRAIL with Ar6pAE2fE3F significantly reduced tumor growth relative to Ar6pAE2fE3F alone. Additionally, use of AGVhTRAIL with Ar6pAE2fE3F increased the number of complete or partial tumor regressions observed at study end. These data provide evidence that coadministration of an oncolytic vector with a gutless vector holds promise for potentiating tumor ablation efficacy.

Optimization of the generation and propagation of gutless adenoviral vectors.

Adenoviral vectors devoid of all viral coding regions are referred to by many names, including gutless vectors. Gutless vectors display reduced toxicity and immunogenicity, increased duration of transgene expression, and increased coding capacity compared to early generation vectors, which contain the majority of the viral backbone genes. However, the production of gutless vectors at a scale and purity suitable for clinical use has limited the utility of this technology. In this work we describe the optimization of the production of gutless vectors. We constructed an improved helper virus and generated an alternative gutless vector producer cell line, PERC6-Cre. We demonstrated increased gutless vector yields, minimal helper virus contamination, and no replication-competent adenovirus contamination using the optimized system. Furthermore, the PERC6-Cre cells were adapted to serum-free suspension culture and high-titer gutless vector preparations were produced using bioreactor technology, suggesting the feasibility of gutless vector scale-up for clinical use. Finally, we observed that helper virus lacking a packaging signal could be packaged at a low frequency, revealing an inherent limitation to the differential packaging strategy for gutless vector propagation.