Development of targeted therapy for a broad spectrum of cancers (pancreatic cancer, ovarian cancer, glioblastoma and HCC) mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences

Background: The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers, while existing at a nearly undetectable level in the surrounding normal tissue. Single promoter vectors expressing diphthe- ria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superfi- cial cell carcinoma of the bladder, pancreatic cancer and ovarian cancer (treated with H19-DTA). However this tar- geted medicine approach may be limited, as not all cancer patients express high levels of H19 and it requires prereq- uisite diagnostic test for H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single con- struct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4. Methods: H19 and IGF2-P4 gene expression was tested in cell lines of a broad spectrum of different carcinomas (bladder, pancreas, ovary, glioblastoma and HCC), by RT-PCR. The therapeutic potential of the double promoter toxin vector H19-DTA-(IGF2)-P4-DTA was tested in the dif- ferent cancer cell lines. Results: The double promoter vector exhibited superior inhibition activity compared to the single promoter expression vectors, in the different cancer cell lines furthermore, the double promoter vector H19- DTA-P4-DTA exhibited augmented-than-additive anti-cancer activity relative to single promoter expression vectors carrying either DTA sequence alone, when tested in a broad spectrum of tumor cells. Conclusions: Our findings show that administration of H19-DTA-P4-DTA has the potential to reach tumor cells, deliver its intracellular toxin without targeting normal tissues, and thus may help reduce tumor burden, improve the quality of life of the patient; and pro- long their life span. As H19 and IGF2 were expressed in a broad spectrum of different cancers, therefore we propose a double promoter expression approach for treating a variety of tumors expressing H19, IGF2, or both. According to this approach patients may be treated with a single double promoter expression toxin vector which is under the con- trol of the IGF2 and H19 regulatory sequences, differentially expressed in those cancers. As majority of the tumor cells express H19, IGF2, or both, therefore the use of prerequisite diagnostic test will be unnecessary.