|Budget Amount *help
¥12,100,000 (Direct Cost: ¥12,100,000)
Fiscal Year 2002: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2001: ¥7,600,000 (Direct Cost: ¥7,600,000)
We previously succeeded overproduction of the HBV env L particles in yeast cells (up to 42% of the total soluble protein). In the present studies, the L particles have been purified, characterized, and examined for the applicability to the gene delivery system. By AFM observation and sedimentation equilibrium, about 110 molecules of L proteins were found to be assembled into a lipid vesicle to form a spherical particle (-500 nm in diameter). To examine the L particles as gene carriers, a mammalian expression plasmid for GFP (green fluorescence protein) was incorporated into L particles by electroporation. 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. Then, the nude mice transplanted with human hepatoma HuH-7 cells and human colon cancer WiDr cells were injected intraperitoneaUy with the L particles containing the plasmid. Two weeks later, the fluorescence was observed specifically in the HuH-7 cells, but neither in the WiDr cells nor in the liver, spleen, kidney, and intestine of the mice. 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. We are now evaluating the effectiveness of L particles as the novel drug delivery system, together with the genetically engineered L particles that can be applied for the pinpoint gene/drug delivery system to different tissues.