Abstract
Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Nanoparticle-based gene delivery technique potentially overcomes these concerns and may be applicable to cancer gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Our previous study demonstrated that an apoptosis inducer, angiotensin II type 2 receptor plasmid DNA (pAT2R) encapsulated in a modified HIV-1 TAT peptide (dTAT-pAT2R), significantly attenuated the growth of Lewis lung carcinoma (LLC) allograft in mouse lungs (Kawabata et al., Cancer Res, 2012). Here, we report a newly synthesized polylysine CPP (K9 peptide)-based gene therapy for lung cancer treatment. The pAT2R and K9 peptide (K9-pAT2R) complexes were condensed using calcium chloride (K9-pAT2R-Ca2). The resulting complexes were small (150 nm) and showed high levels of gene expression in vitro. This simple non-viral formulation approach showed negligible cytotoxicity in several different human and mouse cell lines (human cervix, breast, kidney, and human and mouse lung cell lines). Additionally, this K9-pDNA-Ca2 complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray into LLC orthotopic allograft-bearing mice. Average lung weights (mg) of the K9-pAT2R-Ca2 IT (190.648.3) and the K9-pAT2R-Ca2 IV (201.667.0) treated groups were significantly smaller than that of the control PBS group (325.769.4, P<0.05). In histological examination of tumors in H&E stained lung, average numbers of tumor nodules in the lungs in the K9-pAT2R-Ca2 IT (6.04.2) and the K9-pAT2R-Ca2 IV (4.63.4) groups were also significantly smaller than that of the control PBS group (17.86.0, P<0.01). This tumor attenuating effect was not observed in K9-pLUC-Ca2 IT and K9-pLUC-Ca2 IV treatment, thus it is suggested that LLC tumor growth was attenuated by apoptosis inducer gene, pAT2R, delivery. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth offering preclinical proof of concept for a novel non-viral gene delivery method exhibiting effective lung tumor gene therapy via either IV or IT administration. This work is supported in part by Savara Pharmaceuticals (CB and MT), Faculty of Pharmacy of King Abdulaziz University, Jeddah, Saudi Arabia (NAA), University of Kansas Macromolecule and Vaccine Stabilization Center (CB), Kansas State University Johnson Cancer Research Center (MT), NIH grants U43 CA165462 (MT), P20 GM103418 (MT), and Kansas Bioscience Authority Collaborative Cancer Research grant (MT).