High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression.

Polyethylene glycol (PEG)ylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that acridine-modified lysine (Acr) in (Acr-Lys)(6)-Cys-PEG(5kDa) stabilizes a 1-µg pGL3 dose for up to 1 h in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 µg of pGL3. In this study, we report that increasing the spacing of Acr with either four or five Lys residues markedly increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 h post DNA administration. Co-administration of a decoy dose of 9 µg of non-expressing DNA polyplex with 1 µg of pGL3 polyplex further extended the HD-stimulated expression to 9 h. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 h and provides an understanding as to why polyplexes or lipoplexes prepared with polyethylenimine, chitosan or Lipofectamine are inactive within 5 min following intravenous dosing.

Metabolically stabilized long-circulating PEGylated polyacridine peptide polyplexes mediate hydrodynamically stimulated gene expression in liver.

A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X)(n)-Cys-polyethylene glycol (PEG), possessing 2-6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with PEG (PEG(5000 kDa)) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys)(6)-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 h. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 µg in 50 µl), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5 to 60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 µg of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.