Exploiting the Intron-splicing Mechanism of Insect Cells to Produce Viral Vectors Harboring Toxic Genes for Suicide Gene Therapy

Abstract

Two mammalian introns, the human growth hormone intron and the Simian virus 40 large T antigen intron, were inserted into the coding sequences of diphtheria toxin fragment A (DT-A) and barnase (Bar), respectively, to disrupt their open-reading frames (ORFs). Expression of these two toxic proteins were totally abolished, which enabled the production of normal levels of recombinant baculoviral and adeno-associated viral (AAV) vectors in insect cells. When these viral vectors were introduced into mammalian cells, the introns were spliced out and the toxic proteins were expressed, which resulted in apoptosis in mammalian cells. This is the first report to show that viral vectors harboring toxin genes can be produced at normal levels by exploiting the intron-splicing mechanism of insect cells. Furthermore, viral vectors carrying the DT-A gene under control of tumor-specific promoters were able to exert tumor-specific cell killing. This novel method to produce viral vectors harboring toxic genes under control of tumor-specific promoter offers a powerful tool for further research, as well as for the development of toxin-based suicide gene therapy drugs.