Cancer cell-specific gene disruption of VEGF-A using Cas9

Abstract

Purpose: Angiogenesis inhibitor therapy for cancer is an efficient treatment as a "starvation tactic" against tumor tissues. Angiogenesis inhibitors, such as bevacizumab (avastin), a Vascular endothelial growth factor A (VEGF-A) inhibitor, and ramucirumab, a VEGF-A receptor inhibitor, have already been approved for cancer treatment. However, anti-angiogenic therapy has no specificity for target tissues; thus, angiogenesis inhibitors impede wound healing, causing bleeding and reopening of cuts. In this study, we investigated the possibility of genetic anti-angiogenic therapies that specifically target cancer cells using lentiviral vector-mediated transfection of the Cas9 gene, which is controlled by the promoter region of the growth factor cytokine, midkine. Midkine is a growth factor highly expressed in cancer cells, and its promoter region can be used to selectively regulate gene expression in cancer cells.

Methods: Two lentiviral vectors, one containing the human codon-optimized Cas9 (hCas9) gene and the other containing single guide RNA (sgRNA) sequences targeting VEGF-A, were used to infect human bladder cancer-derived cells.

Results: The gene expression level of hCas9 in bladder cancer cell lines was significantly higher than that in non-malignant cells, and VEGF-A disruption was observed only in bladder cancer cell lines.

Conclusions: These results suggest that placing cas9 under the control of the midkine promoter reduces its expression and activity in non-malignant cells. This approach is thus feasible as a safe anti-angiogenic therapy that does not affect non-target tissues and has few adverse side effects.