Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery

Abstract

PEGylated glycoproteins (PGPs) were synthesized by co-polymerizing a Cys-terminated PEGpeptide, glycopeptide and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20%-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE, and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 hour. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 μg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 hours. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.

Gene Expression in Lung and Liver After Intravenous Infusion of Polyethylenimine Complexes of Sleeping Beauty Transposons

“…Fig. 4B; Chen et al, 2007). In terms of the overall level of expression achieved, we note that the levels of luciferase activity 2 weeks postinfusion were 100-to 1000-fold lower after PEI-mediated delivery compared with hydrodynamic injection under the conditions we employed.…”

“Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years”

“…If the circulating DNA remains intact, gene expression in the liver is equivalent to that achieved by direct hydrodynamic delivery. Delayed hydrodynamic stimulation has been applied to study the circulatory stability of DNA nanoparticles [60][61][62][63]. Biodistribution studies established stable DNA nanoparticles cross the liver fenestrae and transiently move through the space of Disse, prior to hydrodynamic stimulation into hepatocytes [64].…”

“…Disulfide crosslinked peptide DNA polyplexes transfected cells in vitro with efficiency comparable to PEI [95]. A similar formulation was delivered to mice, and was shown to provide some protection to DNA in the circulation for 5 min before delayed hydrodynamic stimulation was performed [60].…”

“…Crosslinking melittin was as efficient as PEI in mediating in vitro transfection [95]. When co-polymerized with a PEG-peptide and glycopeptide, the resulting PEGylated glycoprotein protected DNA in the circulation and produced measurable gene expression in the liver of mice upon hydrodynamic stimulation [60]. In addition, melittin was attached to a polyacridine peptide through either a reducible disulfide linkage or a non-reducible maleimide linkage [220].…”

Efficient reduction of serum cholesterol by combining a liver-targeted gene delivery system with chemically modified apolipoprotein B siRNA

“…Many viral and non-viral vectors (carriers) for liver-targeted gene delivery have been developed for either in vivo or ex vivo/in vitro use. Cationic polymers containing liver-specific molecular ligands (e.g., galactose), which are recognized by membrane receptors [1][2][3], or lipid-based formulations [4,5] have been studied as non-viral vectors for liver-targeted gene delivery. Viral-based vectors for liver-targeted gene delivery are recognized mainly by hepatocyte membrane receptors [6][7][8].…”