In Vivo Gene Transfer Using Sulfhydryl Cross‐Linked PEG‐Peptide/Glycopeptide DNA Co‐Condensates

Abstract

Recent interest in sulfhydryl cross-linked nonviral gene delivery systems, designed to trigger the intracellular release of DNA, has inspired studies to establish their utility in vitro. To determine if this concept can be extrapolated to in vivo gene delivery, sulfhydryl cross-linking peptides (dp 20), derivatized with either an N-glycan or polyethylene glycol (PEG), were used to generate sulfhydryl cross-linked gene formulations. The biodistribution, metabolism, cell-type targeting, and gene expression of sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates were examined following i.v. dosing in mice. Optimal targeting to hepatocytes was achieved by condensing (125)I-DNA with an add-mixture of 10 mol % triantennary glycopeptide, 5 mol % PEG-peptide, and 85 mol % backbone peptide. Four backbone peptides were substituted into the formulation to examine the influence of peptide metabolism and disulfide bond strength on the rate of DNA metabolism and the level of gene expression in vivo. The half-life of DNA in liver was extended from 1 to 3 h using a backbone peptide composed of d-amino acids, whereas substituting penicillamine for cysteine failed to further increase the metabolic stability of DNA. Optimized gene delivery formulations transiently expressed secreted alkaline phosphatase in mouse serum for 12 days. The results suggest that disulfide bond reduction in liver hepatocytes proceeds rapidly, followed by peptide metabolism, ultimately limiting the metabolic half-life of sulfhydryl cross-linked DNA condensates in vivo.

Polymer‐coated polyethylenimine/DNA complexes designed for triggered activation by intracellular reduction

“…Cell-specific delivery of therapeutic genes faces a related challenge: vectors should be stable in the extracellular environment, enabling systemic delivery and targeting of a robust entity to the site of disease, but following arrival within the target cell the vector should release the contained nucleic acid in a form suitable for transcription or translation [5][6][7][8][9][10][11][12].…”

Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery

“…This strategy would allow polymeric peptides to undergo reduction into their monomeric components after cellular internalization (42). We have previously utilized DNA as a template to polymerize either one or two Cys-terminated peptides into polymers (22,31). However, template polymerization becomes inherently more difficult as the number of peptide components increase.…”

“…We have also demonstrated the necessity for PEG to block protein binding and the recognition of DNA condensates by the reticuloendothelial system (10,22,46). Binary peptide copolymers were prepared from glycopeptides and PEG-peptides using plasmid DNA to conduct a template polymerization (22).…”

“…The resulting polypeptide DNA condensates mediate enhanced in vitro gene transfer, presumably by triggered release of the DNA following intracellular reduction of disulfide bonds (33). To test the utility of this approach in vivo, we have reported the use of a Cys-terminated glycopeptide and PEG-peptide that co-polymerize on DNA, direct its biodistribution through receptormediated targeting to hepatocytes in mice, and produce measurable but low-level gene expression in vivo (11,22).…”

Peptides as Promising Non-Viral Vectors for Gene Therapy

“…Upon optimisation, some constructs transfected at levels that were comparable or even higher than LipofectAce, a commercial reagent used as a positive control (McKenzie et al, 2000). Disulfide-linked oligolysine was then further functionalised with triantennary Nglycan signals to target hepatocytes and evaluated in vivo (Kwok et al, 2003). However, contrary to results in vitro, the particles were not stable enough in the reductive intracellular liver environment and premature plasmid release ultimately limited gene expression.…”