AT2R Gene Delivered by Condensed Polylysine Complexes Attenuates Lewis Lung Carcinoma after Intravenous Injection or Intratracheal Spray.
AT2R Gene Delivered by Condensed Polylysine Complexes Attenuates Lewis Lung Carcinoma after Intravenous Injection or Intratracheal Spray.
复制标题
DOI：
10.1158/1535-7163.mct-15-0448
复制
发表时间：
2016-01
影响因子：
5.7
通讯作者：
Tamura M
中科院分区：
文献类型：
--
作者：
Alhakamy NA;Ishiguro S;Uppalapati D;Berkland CJ;Tamura M
转染效率和毒性问题仍然是基因治疗的挑战。细胞穿透肽 (CPP) 已被广泛研究以改善遗传物质（例如 pDNA 和 siRNA）的转染。在此，将合成的 CPP（多聚赖氨酸，K9 肽）与血管紧张素 II 2 型受体 (AT2R) 质粒 DNA (pAT2R) 复合，并使用氯化钙浓缩复合物。所得复合物很小（~150 nm），并且在体外和体内表现出高水平的基因表达。这种简单的非病毒制剂方法在四种不同的人类细胞系（子宫颈、乳腺癌、肾和肺细胞系）和一种小鼠细胞系（肺癌细胞系）中显示出可忽略不计的细胞毒性。此外，当通过静脉内 (IV) 注射或气管内 (IT) 喷雾给药时，这种 K9-pDNA-Ca2+ 复合物证明了癌症靶向基因传递。在体外培养的 Lewis 肺癌 (LLC) 细胞系和同基因小鼠的原位癌症移植物中评估了转染效率。免疫组织化学分析证实，该复合物有效地将 pAT2R 递送至癌细胞，并主要在癌细胞和支气管上皮细胞中表达。这些复合物的单次给药显着减弱了肺癌的生长，为新型非病毒基因递送方法提供了临床前概念证明，该方法通过 IV 或 IT 给药表现出有效的肺肿瘤基因治疗。
Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Here, a synthetic CPP (polylysine, K9 peptide) was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R) and complexes were condensed using calcium chloride. The resulting complexes were small (~150 nm) and showed high levels of gene expression in vitro and in vivo. This simple non-viral formulation approach showed negligible cytotoxicity in four different human cell lines (cervix, breast, kidney, and lung cell lines) and one mouse cell line (a lung cancer cell line). Additionally, this K9-pDNA-Ca2+ complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray. The transfection efficiency was evaluated in Lewis lung carcinoma (LLC) cell lines cultured in vitro and in orthotopic cancer grafts in syngeneic mice. 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.