|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2004 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2003 : ¥2,200,000 (Direct Cost : ¥2,200,000)
We have already reported a novel vector, consisting of bloc copolymer ; polyethylene glycol and poly-L-lysine. The DNA complex had core-shell structures, yielding spherical nano-particles, polyion complex (PIC) micelle, which enabled DNA to stay intact molecule for six hours. PIC micelle also enabled in vitro and in vivo gene transfer, but the efficiency of expression was not enough.
In this project, we report a novel block copolymer based PIC, particularly available for in vivo gene transfer.
2.Materials and Methods
Polyaminoethylene aminopropyl aspartamide was used for in vitro and in vivo expression studies. For in vitro gene transfer, PIC micelles were incubated with cells for 24 hr, followed by further 48 hr incubation. The cells were collected, lysed and subjected to measure luciferase activity. For in vivo gene transfer, the PIC micelles were sprayed in the trachea by a spray gun. The lung was analyzed for luciferase activity.
3.Results and discussion
PIC micelles with PEG-DET were tested for in vitro and in vivo gene transfection. HepG2 cells transfected with the micelles with higher charge ratio showed higher gene expression. The polymerization degree did not affect gene expression significantly. The same tendency was obtained in Cos-1 cells, vascular endothelial cells, and THP-1 cells.
For in vivo gene transfer, PIC micelles with polymerization degree of DET segment 68 and charge ratio of 1:80 was used. After 1 day of transfection, the lung had very high levels of luciferase activity, 40 times higher than that of polyethyleneimine. The expression increased until 3days after the transfection, and lasted for 14 days. PIC micelles were injected into the left ventricle of apolipoprotein E knockout mouse. Gene expression was observed only in the aorta and not in other organs. These results suggest that PEG-DET is useful for vectors of gene therapy.