Preclinical gene therapy studies using the Sleeping Beauty gene delivery system

Nonviral gene therapy vectors show promise for human gene therapy. However, nonviral vectors generally suffer from transient gene expression and poor gene transfer efficiency relative to viral vectors. To overcome this problem we used the Sleeping Beauty (SB) transposable element to achieve transgene integration and long-term expression in animal models of hemophilia A and glioblastoma. Hemophilia A mice are deficient in coagulation factor VIII (FVIII) and mount and immune response to FVIII, thereby recapitulating the human clotting dysfunction and immune complications. We tolerized hemophilia A mice to FVIII by injecting FVIII protein immediately after birth to prevent anti-FVIII antibody formation. Tolerized hemophilia A mice were phenotypically corrected of clotting dysfunction by administering FVIII-expression transposons and SB transposase-encoding plasmid DNA by high-pressure tail vein injection. In addition, we employed the SB gene delivery system as a vector for cancer gene therapy against human glioblastoma xenografts. Transposons were delivered to glioma xenografts by intratumoral injection of DNA/polyethylenimine (PEI) complexes. After six intratumoral injections, roughly 8% of a glioma xenograft could be stably transfected by SB-mediated gene transfer. Using this gene delivery approach, we showed that delivering anti-angiogenic or cytotoxic genes into glioma xenografts has a marked anti-tumor effect. 50% of mice treated with SB vectors encoding an angiostatin-endostatin fusion gene and a soluble vascular endothelial growth factor receptor were rendered tumor-free after two intratumoral injections. This is the first demonstration that a transposable element can be used to deliver anti-angiogenic genes for cancer therapy. Since gliomas overexpress the IL-13 receptor, we created an IL-13 fusion protein to selectively kill glioma cells; where IL-13 was fused to the known suicide gene, HSV-TK. Conditioned media containing the IL-13-HSV-TK fusion killed glioma cells in an IL-13-receptor dependent manner. 60% of xenografts injected with an IL-13:HSV-TK fusion gene were cured after ganciclovir administration, and tumors treated with the fusion were highly infiltrated with macrophages. Thus, IL-13:HSV-TK is a novel anti-tumor protein that kills tumors in a targeted manner by ganciclovir-mediated death and also by potentiating anti-tumor immunity. In summary, these studies show that SIB is a nonviral vector that shows potential for use in human gene therapy.