Effective Transgene Constructs to Enhance Gene Therapy with Trichostatin A

Abstract

Gene therapy has been proposed as a strategy for the treatment of intractable human diseases since the early 1990s. For the expression of a specific transgene in desired cells or tissues with the proper timing, many vectors carrying transgenes have been developed (Matrai et al., 2010; Nayak & Herzog, 2010; Sliva & Schnierle, 2010). Retroviral, lentiviral, adenoviral, and adeno-associated viral vectors are used in various ways to achieve these goals. However, the introduced transgenes frequently become silenced in the host cells (Harbers et al., 1981; Jahner et al., 1982; Palmer et al., 1991). We searched for bioactive substances from Actinomycetes that enhance transgene expression, and found that trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), enhanced several promoter activities remarkably (Y. Ma et al., 2009). HDACis have been used as anti-cancer drugs, because they have various effects on tumor cells to arrest cell growth, induce apoptosis, inhibit metastasis, and enhance anti-tumor immunity, by regulating the expression of the relevant genes (Bolden et al., 2006; Haberland et al. 2009; X. Ma et al., 2009; Mai et al., 2005). Here, we developed effective TSA-inducible killer constructs to enhance the anti-cancer effects of TSA, by identifying the TSA-responsive element of the herpes simplex virus thymidine kinase (hsvTK) promoter, and TSA-dependently activating some cell-death-inducing genes. We determined the most relevant regions responsive to TSA, and constructed chimeric promoters with higher fold-increases and greater induced strengths with TSA, by replacing the weak TSA-responsible region (TSA2) of the CMV promoter with two or three copies of the TSA-responsible sites (TSA1) of the hsvTK promoter. In addition, the synthetic intron sequence (0.2kb) from the pRL-TK vector and the long 3’-untranslated region (1.0kb) from the pSV2-neo vector, including the SV40 late polyA site, were important for the basal expression of the transgene and the TSA-induction, respectively. To create the TSA-inducible killer constructs, we placed the hsvTK gene for combination therapy with the prodrug Ganciclovir, and some strong death-inducing molecules (Bax, caspase8, and TRIF) under the control of the TSA-responsible chimeric promoters. They effectively killed the cells in which they were introduced, in a TSA-dependent manner. To evaluate the utility of the killer constructs for cancer gene therapy, the TSA-dependent death-inducing constructs were transferred to retroviral and adenoviral vectors.