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

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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.