Although the combination of gene therapy and virotherapy for cancer therapy has obtained some encouraging results in vitro and in vivo over the past few years, some improvements of the vectors are still urgently needed to enhance their therapeutic effects for cancers. In order to maximize the anti-cancer activities of conditionally replicating adenoviral vectors (CRAd) vector, we for the first time generated a novel CRAd vector by inserting an expression cassette between E4 and the fiber using homologous recombination and tested this vector in melanoma cancer therapy. By using this novel vector we expressed two therapeutic transgenes, IL-24 and arresten, inserted in E1 and the region between E4 and the fiber, respectively, to test the melanoma inhibitory activities of this oncolytic virus in vitro and in vivo. The two therapeutic transgenes were successfully expressed by the novel CRAd, which was confirmed by western blotting. We then showed that this novel CRAd vector significantly suppressed the tumor growth of melanoma in vitro and in vivo compared with the control by inducing apoptosis and inhibiting angiogenesis. The novel CRAd vector generated in this study holds promise for developing more effective therapeutics for not only melanoma but also other common cancers.
Homologous recombination (HR) is a strategy for genetic correction. The efficiency of HR can be increased by creating a targeted double-strand break (DSB) via zinc-finger nucleases (ZFNs) and/or by introducing linear donor DNA intracellularly. Some studies have suggested that increased copy numbers of linear donor DNA may further improve HR efficiency. However, the introduction of multiple copies of a linear donor fragment remains a challenge, particularly in cell types with low transfection efficiency. In this study, we developed a vector that carries tandem repeats of a donor fragment, with each repeat flanked by ZFN target sequence fragments (TSFs). The cleavage of the flanking TSF sequence by ZFN would lead to the release of multiple linear fragment. We demonstrated that this novel vector carrying five copies of a linearizable donor fragment, when co-transfected with a ZFN-expressing vector in 293 cells, showed improved HR efficiency about 30 times, as compared with vector carry nonlinearizable donor. For the application of gene therapy, we then introduced this system into an adenoviral vector, which also revealed markedly improved ZFN-mediated HR efficiency in cells. The novel strategies presented here have the potential to promote the application of ZFNs in both basic research and disease therapy.
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