Development of a Sleeping Beauty-based telomerase gene delivery system for hepatocytes.

Telomerase is a particular reverse transcriptase that not only synthesizes and maintains the telomere but also promotes the proliferation of resting cells and prevents cellular senescence. The advantages of the Sleeping Beauty transposon system include prolonged transgene expression without eliciting an immunogenic response, no possibility of RCV and ease of construction. Tissue-specific therapeutic gene expression is extremely important in gene therapy, because non-specific expression can cause an immune response of the transduced cells that can severely limit the stability of the transgene. The SB system containing the telomerase gene controlled by two chimeric transthyretin (TTR) gene promoters/enhancers, the human alcohol dehydrogenase gene promoter (ADHp), and the SV40 viral enhancer (SV40VE) was constructed in order to activate hepatocyte cell growth. The higher expression was achieved using these elements and FACS analysis showed that this system was effective in hepatocyte targeted gene therapy. Our new SB mediated telomerase delivery system for hepatocytes can be used in human gene therapy applications.

Sleeping Beauty-mediated suicide gene therapy of hepatocellular carcinoma.

The aim of this study was to use gene therapy via the Sleeping Beauty (SB) system to increase telomerase promoter activity to target hepatocellular carcinoma (HCC). In previous studies, we identified selective and increased expression of luciferase and suicide genes controlled by the hTERT (human telomerase reverse transcriptase) promoter and the SV40 enhancer in telomerase-positive cancer cell lines. Because telomerase is activated in about 80% of HCCs, it is likely that increasing the activity of the telomerase promoter with a suicide gene will effectively eradicate HCCs. We found that the telomerase promoter mediated SB system can efficiently insert transgene into HCC genomes. Also, telomerase promoter activity was increased using a SB vector expressing suicide gene HSV-TK (herpes simplex virus thymidine kinase) controlled by the hTERT promoter and a SV40 enhancer for the induction of telomerase-positive cancer-specific cell death. HCC cell lines transfected with pT.hTp.HSV-tk.Con with active helper plasmid and ganciclovir (GCV) significantly inhibited cancer cell growth. These results indicate that Sleeping Beauty transposon mediated suicide gene expression can be used in HCC-targeted cancer gene therapy.

Protection from acute cellular injury using Sleeping Beauty mediated telomerase gene transfer.

We developed a Sleeping Beauty (SB) transposon mediated hTERT gene delivery system for in vitro use. We have constructed telomerase or luciferase gene expressing SB-transposons with a SV40 enhancer (pT3.hTERT.Con and pT3.Con, respectively) or without an enhancer (pT3.Pro). Using the SB transposon system in vitro hTERT gene overexpression has protective effects from acute cellular injury by tert-butyl hydroperoxide (t-BH), carbon tetrachloride (CCl(4)), and d-galactosamine (d-GalN) in normal human cells IMR-90. pT3.hTERT.Con vector and helper plasmid co-transfection resulted in a approximately 3-fold increase in telomerase activity which was maintained for 14 days. Trypan blue and Cell Death Detection Assays showed the protective effects of the telomerase gene against toxic agents. Fourteen days after co-transfection with pT3.hTERT.Con vector and helper plasmid, IMR-90 cells were incubated with 1.2mM t-BH for 50 min, 5mM CCl(4) for 1.5h or 30 mM d-GalN for 24h. Cell viability of SB-mediated telomerase overexpressing cells significantly increased by 48% (t-BH), 43% (CCl(4)), and 25% (d-GalN) in comparison to mock treated cells. Cell Death Detection ELISA showed a decrease in the rate of apoptosis by 47%. In summary, SB transposon mediated telomerase gene transfer may have a protective effect against t-BH, CCl(4), or d-GalN induced acute cellular injury, and this results suggested SB-mediated telomerase therapy for tissue engineering.

Enhanced expression of apoptin by the Myc-Max binding motif and SV40 enhancer for SCLC gene therapy.

Apoptin is derived from chicken anemia virus (CAV) and known to induce tumor specific apoptosis but not normal cells. The aim of this study was to use increased expression of apoptin by the Myc-Max response element (MMRE) and SV40 enhancer in small-cell lung cancer (SCLC) gene therapy. To investigate the possibility of the utilization of the MMRE, apoptin, and SV40 promoter/enhancer in targeted cancer gene therapy, adenovirus vector expressing apoptin controlled by the MMRE, and SV40 promoter/enhancer was constructed. Ad-MMRE-apoptin-enh infected SCLC cells were significantly suppressed and induced apoptosis more than those of Ad-apoptin or Ad-apoptin-enh. Infection with Ad-MMRE-apoptin-enh of normal cells did not increase apoptosis. About 85% of SCLC tumors show overexpression of the myc family, so the increased expression of apoptin by MMRE and SV40 enhancer can be used in targeted SCLC gene therapy. These results indicate that apoptin expression was increased by the MMRE and SV40 promoter/enhancer, and that this strategy can be used in SCLC targeted cancer gene therapy.

Adenovirus-mediated suicide SCLC gene therapy using the increased activity of the hTERT promoter by the MMRE and SV40 enhancer.

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc-Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60 approximately 80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer.

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.

Adenovirus-mediated HSV-TK gene therapy using the human telomerase promoter induced apoptosis of small cell lung cancer cell line.

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)n to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, but not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the place of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into a normal and a small cell lung cancer cell line. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, cell death detection ELISA and FACS analysis suggest that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for SCLC gene therapy.

Adenovirus-mediated suicide gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter induced apoptosis of ovarian cancer cell line.

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)(n) to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, yet not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We here tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the replace of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into normal and human gynecological cancer cell lines. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, FACS analysis and TUNEL assay suggests that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for gynecological cancer gene therapy.