Development of novel gene-therapy using IL-12 and DNA nanoassociate bearing hyaluronan-glycocalyx for recurrent hepatocellular carcinoma after liver transplantation

在本项目中，我们研究了IL-12和DNA纳米关联透明质酸-糖基化修饰肝窦内皮细胞对肝移植后复发肝细胞癌的基因治疗效果。首先，对mIL-12质粒DNA进行纯化。为了建立一种将外源DNA靶向SECS的体系，通过将HA偶联到聚L赖氨酸(PLL)上，制备了含有HA侧链的梳状聚阳离子。将得到的HA接枝-PLL共聚物(PLL-g-HA)与DNA在154 mM的氯化钠溶液中混合，形成含有水化透明质酸盐壳的可溶纳米缔合物。琼脂糖凝胶滞留分析表明，尽管存在多阴离子HA侧链，PLL-g-HA仍与DNA选择性相互作用。证实了PLL-g-HA与mIL-12质粒DNA的聚合。然而，由于缺乏PLL-g-HA，PLL-g-HA/pCAGGS-mIL-12的稳定生产和供应是困难的。C3H小鼠(H-2(K))为S.C.接种2.5×10(6)MH134细胞(H-2(K))，用mIL-12质粒DNA对已建立的肝癌进行电穿孔基因治疗。瘤内mIL-12、干扰素-γ和干扰素-γ诱导蛋白-10基因转移显著抑制肝癌的生长，与肝癌转移对照pCAGGS质粒相比。

In this project, we investigated the effectiveness of gene therapy using IL-12 and DNA nanoassociate bearing hyaluronan-glycocalyx argeting the sinusoidal endothelial cells for the treatment of recurrent hepatocellular carcinoma after liver transplantation. First of all, mIL-12 plasmid DNA was purified. To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains.PLL-g-HA. Polymerization of PLL-g-HA with mIL-12 plasmid DNA was then confirmed. However, stable production and supply of PLL-g-HA/pCAGGS-mIL-12 was difficult due to the lack of PLL-g-HA. C3H mice (H-2(k)) were s.c. implanted with 2.5 x 10(6) MH134 cells (H-2(k)) and we treated the established HCC with electroporation-mediated gene therapy using mIL-12 plasmid DNA. Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-gamma, and IFN-gamma-inducible protein-10, significantly inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid.