Targeted Delivery of DNA Using YEE(GalNAcAH)3, a Synthetic Glycopeptide Ligand for the Asialoglycoprotein Receptor

Abstract

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.

Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery

“…[11][12][13][14] Selective uptake by a broad range of cell types could be induced by incorporating homing devices for cell-specific receptors. 9,10,15,16 Polyethyleneimine (PEI) [17][18][19] and dendrimer polyamido amines 20,21 gave even higher transfection yields. The high transfection efficacy was explained by the intrinsic endosomolytic activity of these polymers, facilitating the escape of the DNA cargo into the cytosol.…”

In vivo targeted gene transfer into liver cells mediated by a novel galactosyl-D-lysine/D-serine copolymer

“…[19][20][21] In other studies, animals were pretreated with colchicine and chloroquine to obtain high expression levels of transgene. 6 In contrast, we observed efficient expression of reporter genes in mouse liver without prior surgical procedures or drug treatment.…”

“…In addition, it is clear that preparation of DNA-ligand complexes remains a significant technical obstacle, and improved methods will likely be required To date, a variety of DNA-ligand complexes have been developed. [3][4][5][6][7][8][9] Polycations and proteins that carry numerous positive charges electrostatically bind polyanionic DNA to form condensed molecular aggregates.10 Polylysine is the most commonly used DNA binding moiety; it has an electrostatic affinity with DNA, and the lysine ⑀-amino groups can be covalently bound to ligand molecules. On the other hand, DNA-polylysine complexes formed in physiological saline tend to aggregate or precipitate due to neutralization of the charges.…”

Glycobiology in Medicine

“…The conjugate is then ionically complexed with a polynucleotide, and injected into the blood stream. Later designs utilize small chemi cally synthesized ligands instead of desialylated al-acid glycoprotein [47,88], These molecules appear to be as effective as desialylated al-acid glycoprotein. As an alter native, the amino groups of polylysine can be partially modified with a simple sugar (e.g.…”