Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides - Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor

Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleot ide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser(365) position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and n egatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stabil ity and efficiency in targeting hepatocytes via the asialoglycoprotein rece ptor. Physical characterization and electron microscopy revealed that the o ligonucleotides were efficiently encapsulated within the liposomes, with th e positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lacto sylated polyethyleneimine provided the most intense nuclear fluorescence wi th the fluorescein-labeled oligonucleotides. The lactosylated polyethylenei mine and the three different liposomal formulations resulted in A to C conv ersion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatoc ytes. The results suggest that both the liposomal and polyethyleneimine for mulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the int roduction or repair of genetic mutations by the chimeric RNA/DNA oligonucle otides.