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 nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the 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 lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.
Bandyopadhyay
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P
Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Ma
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Linehan-Stieers
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C
Kren
B T
BT
Steer
C J
CJ
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D016503:Drug Delivery Systems; D005164:Factor IX / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D008081:Liposomes; D008099:Liver / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism*; D012313:RNA / Q000008:administration & dosage* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012694:Serine / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D008081:Liposomes; D009841:Oligonucleotides; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012694:Serine; D012313:RNA; D005164:Factor IX; D011094:Polyethyleneimine; D004247:DNA