Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells

A. G. Ziady, T. Ferkol, T. Gerken, D. V. Dawson, D. H. Perlmutter, P. B. Davis

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugated was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 ± 0.5 nm in diameter and gave peak transgene expression of 3-4 x 106 ILU/mg which persisted (> 7 x 105 ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 ± 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.

Original languageEnglish
Pages (from-to)1685-1697
Number of pages13
JournalGene therapy
Volume5
Issue number12
DOIs
StatePublished - 1998

Keywords

  • Gene therapy
  • Poly-L-lysine
  • Receptor-mediated gene transfer
  • Serpin enzyme complex receptor
  • Structure-function relationship

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