Novel Strategy of Tumor Gene Therapy by Very Small Plasmid Complex Particles

人们一直致力于开发非病毒载体系统作为病毒的更安全的替代品。但这些人工载体的体内基因表达受到严格限制。主要的障碍应该是复合物与生物组分的不利相互作用，以及复合物的尺寸太大而不能递送到靶细胞。我们在复合颗粒上开发了一种阴离子聚合物涂层，它使颗粒重新充电到负电荷，并有效地减少了非特异性相互作用。然后将PEI与编码细胞因子或病毒特异性蛋白的质粒制备小质粒复合物，并探索其对荷瘤小鼠的抗癌治疗潜力。肿瘤内注射后对小鼠有明显的治疗作用，并获得了较高的治疗效果。为了诱导对肿瘤细胞的更高的免疫应答，应在肿瘤细胞表面有效表达致病性抗原。本研究以腺病毒死亡蛋白（ADP）和结核分枝杆菌早期分泌抗原靶点-6（ESAT-6）两种致病蛋白以及结核分枝杆菌主要分泌蛋白抗原85 B（Ag 85 B）作为免疫应答诱导抗原，将表达细胞因子和致病性抗原的质粒同时转染肿瘤细胞，有望产生高效的抗肿瘤免疫增强作用。由携带致病蛋白基因的质粒形成的小复合物显示出高效的抗肿瘤活性，并且正如预期的那样，这些致病基因与精氨酸基因共转染在荷瘤小鼠中诱导了更高的抗肿瘤治疗效果。对犬原发性肿瘤进行了动物临床实验，观察到明显的抑制肿瘤生长的作用。

Great efforts have been focused on developing the non-viral vector systems as safer alternatives to viruses. But the in vivo gene expression by those artificial vectors is strictly limited. The major obstacles should be adverse interactions of the complex with biocomponents, and the too large size of the complexes to be delivered to the target cells. We developed an anionic polymer-coating on the complex particles, which re-charged the particles to negative, and effectively diminished the non-specific interactions. It also enabled the preparation of concentrated very small DNA complex suspension under the precise conditions.Small plasmid complexes were then prepared with the PEI and the plasmids encoding cytokines or virus specific proteins, and explored for anticancer therapeutic potential with tumor-bearing mice. They showed significant therapeutic effect in mice after intratumoral injection, and fairly high therapeutic effect was obtained.In order to induce higher immune response to tumor cells, expression of the pathogenic antigen on the tumor cell surface should be effective. Simultaneous transfection with plasmids expressing cytokines and pathogenic antigen into tumor cells should, thus, be expected to lead to highly effective anti-tumor immune-enhancement.We employed two pathogenic proteins, adenovirus death protein(ADP) and mycobacterium tuberculosis early secretory antigenic target-6 protein(ESAT-6), and mycobacterium tuberculosis major secretory protein antigen 85B(Ag85B) as an immune response-inducing antigen. The small complexes made of the plasmids harboring the pathogenic protein genes showed highly effective anti-tumor activity, and as expected, co-transfection of those pathogenic genes with cytokine-genes induced much higher anti-tumor therapeutic effect in tumor-bearing mice. Animal clinical study on primary tumor-bearing dogs was also carried out, and evident suppression of the tumor growth was observed.