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Bionanoparticles for Pinpoint Delivery of Genes and Drugs

Akihiko Kondo, Masaru Muraoka, and Hideki Fukuda. Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe, 657-8501, Japan

Gene therapy is recognized as one of the most promising cures for many diseases such as cancer. Many attempts using virus vectors have been made for delivering genes to various cells in human. While these gene therapies have shown noticeable efficacy, it has turned out that nonspecific introduction of genes into undesired cells and organs causes deleterious side effects. More importantly, the virus vector-derived DNA may induce unexpected effects on human. Hepatitis B virus (HBV) is a human liver-specific DNA virus, whose genome harbors three overlapping envelope (env) genes in a single open reading frame, encoding S, M (pre-S2 + S), and L (pre-S1 + pre-S2 + S) proteins. In the last decade, the recombinant HBV env S and/or proteins were produced in yeast cells as particles and used as the immunogen for the new generation HB vaccines that were proven to be safely applicable to human. We previously succeeded overproduction of the HBV env L particles in yeast cells. In the present studies, the L particles have been purified, characterized, and examined for the applicability to the gene delivery system. To examine the L particles as gene carriers, a mammalian expression plasmid for GFP (green fluorescence protein) was incorporated into L particles. The L particles containing the plasmid were added to the culture medium of human hepatoma HepG2 cells. After two days, more than 90% of the HepG2 cells expressed GFP, while the control non-human liver cells did not. Because the L particle is an empty vesicle containing no viral DNA, it can be used as a safe and efficient vector for human liver-specific gene transfer. Genetically engineered L particles that are able to target to various organs were constructed by deleting the hepatocyte binding domain of L protein (pre-S region) and displaying targeting peptide or protein ligands such as dimmer of Z domain (ZZ domain) and single chain variable fragment (scFv), etc. In the ZZ domain displaying particles, displayed ZZ bound antibodies, and hence particles showed the antibody mediated specificity. We have confirmed the antibody-mediated introduction of calcein into target cells