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

We have targeted the serpin enzyme complex receptor for gene transfer in hu man hepatoma cell lines using peptides 30 amino acids in length which conta in the five amino acid recognition sequence for this receptor, coupled to p oly K of 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 magn etic resonance spectroscopy Conjugates were prepared ii, which 3.5%, 7.8% o r 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of th ese conjugates was then coupled with ligand peptides so that one In 370, on e in 1039, or one in 5882 lysines were substituted with receptor ligand. El ectron microscopy and atomic force microscopy were used to assess complex s tructure and size. HuH7 human hepatoma cells were transfected with complexe s of these conjugates with the plasmid pGL3 and luciferase expression measu red 2 to 16 days after treatment. All the protein conjugates in which 26% o f the K residues were modified with sulfo-LC SPDP were poor gene transfer r eagents. Complexes containing less substituted poly K, averaged 17+/- 0.5 n m in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg whic h persisted (>7x 10(5) 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 conjug ates. As few as eight to I I ligands per complex are optimal for DNA delive ry via the SEC receptor The extent of substitution of receptor-mediated gen e transfer complexes affects the size of the complexes, as well as the inte nsity and duration of transgene expression. These observations may permit t ailoring of complex construction for the usage required.