| Recombinant DNA vectors are fundamental tools in gene therapy research. A novel cloning system, pLinus, was made to facilitate vector construction by providing 32 unique restriction sites to adapt DNA fragments in a single step.; To compensate the low delivery efficiency of the non-viral vector systems, we have constructed two high expression plasmid vectors, pHi1/2, by incorporating a transcriptional amplifier strategy into a single construct. In both pHi1/2 vectors, the amplifier expression cassettes contained two independent transcriptional units. One transcriptional unit contained a transcriptional factor, the tat gene, driven by a strong constitutive CMV promoter. The second transcriptional unit contained either an HIV1 LTR or HIV2 LTR driving the gene of interest. Using the human IL-2 cytokine as a reporter and therapeutic gene, the pHi1/2 amplifier vectors could achieve significantly higher IL-2 expression levels than that observed when using the CMV promoter alone. In vivo injection of the stable pHi2-IL-2 gene modified Lewis Lung (LL/2) tumor clones resulted in slower tumor growth and longer survival as compared to those mice injected with either CMV-driven IL-2 transfected clones or the parental tumor cells.; To solve the safety concern, we constructed a novel plasmid vector, pHi-Hot, by combining inducible and amplifier strategies in a single vector. In pHi-Hot, the first transcriptional unit contained an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. Using the human IL-2 cytokine gene as a reporter gene, we demonstrated that, heat shock at 42°C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were significantly higher than achieved by using the hsp promoter or CMV promoter directly. And repeated heat shock at 42°C for 30 min of mice injected with a pHi-Hot-IL-2 gene modified LL/2 clone led to tumor regression.; In this study, three major approaches towards facilitating vector construction and improving vector expression cassette design are described. |
