Chieko Yoshihara

DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice

The highest barriers for non-viral vectors to an efficient in vivo gene transfection would be (1) non-specific interaction with biological molecules, and (2) large size of the DNA complex particles. Protective coating of the DNA/polyethyleneimine (PEI) complexes by hyaluronic acid (HA) effectively diminished the adverse interactions with biological molecules. Here we found HA also protected the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (<70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume. In vivo gene expression efficiency of the small complex was examined with mice subcutaneously inoculated with B16 melanoma cells. These formulations showed high reporter-gene expression level in tumor after intravenous injection into tumor-bearing mice. Small complex was then made of the plasmid encoding GM-CSF gene, and injected into the mice bearing subcutaneous solid B16 tumor. After intravenous injection, it induced apparent tumor growth suppression in 50% of the mice. Notably, significant therapeutic effect was detected in the mice that received intratumoral injection, and 75% of the mice were completely cured with disappearance of tumor.

Highly efficient in vivo gene transfection by plasmid/PEI complexes coated by anionic PEG derivatives bearing carboxyl groups and RGD peptide

A new class of an anionic poly (ethylene glycol) derivative, PEG-Suc, bearing 17.7 pairs of carboxylic acid-side chains was synthesized. PEG-Suc deposited onto the DNA/polyethyleneimine complexes without destroying them even at high dose ratio. Coating of the DNA complexes by PEG-Suc recharged their surface to negative, and effectively protected them from the albumin-induced aggregation. Paired carboxyl groups in the side chains showed higher proton sponge effect. Negatively charged surface would diminish the electrostatic binding of the complexes to the cells, and the transfection efficiency on the cultured cells was not high. RGD peptide side chain as a ligand to malignant cell surfaces was then introduced to compensate the reduced electrical adhesion. RGD-PEG-Suc-coated plasmid/PEI complex brought about more than 3 times higher reporter protein activity on the cultured B16 cells. Those bio-compatible DNA complexes with ligand attained very high gene expression in tumor, lung, and liver after injection into mouse tail vein.

Loosening of DNA/Polycation Complexes by Synthetic Polyampholyte to Improve the Transcription Efficiency: Effect of Charge Balance in the Polyampholyte

High mobililty group proteins are amphoteric nuclear proteins that are known to unfold chromatin to stimulate transcription. To mimic their structures, we synthesized the novel polyethylene glycol (PEG) derivatives, PEG-ACs, consisting of both amino- and carboxyl-pendants in various ratios, and their loosening and transcription-improving activity on the DNA complex was examined. Fluorescence anisotropy measurement revealed that anionic PEG-ACs with more carboxyls than amines could efficiently loosen the DNA/polyethyleneimine complex. Those anionic PEG-ACs showing a loosening effect on the DNA complex evidently increased the transcription rate to >20 times higher than that of the original complex, probably owing to the facilitated approach of transcriptional factors to the DNA segments in the loosened complexes. The complexes with anionic PEG-ACs also showed improved transgene expression level on the cultured cells, indicating the effectiveness of improving transcriptional activity to attain a high extragene expression by the plasmid complex. The loosening mechanism of DNA/polycation complexes was investigated with a simplified model via Monte Carlo simulation to discern the difference in the presence of cationic polyampholytes, anionic polyampholytes, and polyanions.

Antitumor effect of chondroitin sulfate‐coated ternary granulocyte macrophage‐colony‐stimulating factor plasmid complex for ovarian cancer

Although replication‐competent viruses have been developed for treating cancers, their cytotoxic effects are insufficient as a result of infection inhibited by the generation of neutralizing antibodies, and systemic administration is difficult as a result of the life‐threatening serious side‐effects of virus‐induced cytokine surge. To overcome these critical problems, we devised a plasmid/polycation/polyanion complex and assessed the potential of ternary plasmid complexes coated with chondroitin sulfate in gene therapy for ovarian cancer. The antitumor effects of chondroitin sulfate‐coated complex as an anionic component were compared with those of hyaluronic acid on ovarian cancer.

Novel Antitumor Strategy Utilizing a Plasmid Expressing a Mycobacterium tuberculosis Antigen as a “Danger Signal” to Block Immune Escape of Tumor Cells

Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB). This induces presentation of the TB antigen on tumor cell surfaces, which can be recognized by antigen presenting cells (APCs) as a “danger signal” to stimulate antitumor immune response. This strategy is also expected to amplify the immune response against tumor-associated antigens, and block immune escape of the tumor. DNA/PEI/chondroitin sulfate ternary complex is a highly effective non-viral gene vector system for in vivo transfection. A therapeutic complex was prepared using a plasmid encoding the TB antigen, early secretory antigenic target-6 (ESAT-6). This was injected intratumorally into syngeneic tumor-bearing mice, and induced significant tumor growth suppression comparable to or higher than similar complexes expressing cytokines such as interleukin-2 (IL-2) and interleukin-12 (IL-12). Co-transfection of the cytokine-genes and the ESAT-6-gene enhanced the antitumor efficacy of either treatment alone. In addition, complete tumor regression was achieved with the combination of ESAT-6 and IL-2 genes.