Targeted delivery of DNA using YEE(GalNAcAH)3, a synthetic glycopeptide ligand for the asialoglycoprotein receptor.
In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.
Merwin
J R
JR
TargeTech, Inc., Meriden, Connecticut 06450.
Noell
G S
GS
Thomas
W L
WL
Chiou
H C
HC
DeRome
M E
ME
McKee
T D
TD
Spitalny
G L
GL
Findeis
M A
MA
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D004337:Drug Carriers; D018390:Gene Targeting; D006020:Glycopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004337:Drug Carriers; D006020:Glycopeptides; D007457:Iodine Radioisotopes; D009961:Orosomucoid; D011956:Receptors, Cell Surface; C017030:asialoorosomucoid; C092360:tyrosyl(glutamyl)glutamate tris(N-acetylgalactosamineaminohexylglycoside)amide; D011107:Polylysine; D004247:DNA; D008156:Luciferases