Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.

TitleSynthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.
Publication TypeJournal Article
Year of Publication2000
AuthorsAnwer K, Logan M, Tagliaferri F, Wadhwa M, Monera O, Tung CH, Chen W, Leonard P, French M, Proctor B, Wilson E, Singhal A, Rolland A
JournalPharm Res
Volume17
Issue4
Pagination451-9
Date Published2000 Apr
ISSN0724-8741
KeywordsAmino Acid Sequence, Animals, Cells, Cultured, Chloramphenicol O-Acetyltransferase, Gene Expression, Gene Targeting, Glycopeptides, Liver, Mice, Molecular Sequence Data, Plasmids, Rats, Rats, Sprague-Dawley
Abstract

PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration.

METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively.

RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery.

CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.

DOI10.1023/a:1007533121682
Alternate JournalPharm Res
PubMed ID10870990
Related Institute: 
Molecular Imaging Innovations Institute (MI3)

Weill Cornell Medicine
Department of Radiology
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