Non-viral gene therapy offers the potential to deliver nucleic acids producing therapeutic proteins to treat genetic diseases without the limitations observed with viral vectors. Before the therapeutic potential of non-viral gene delivery can be realized, several barriers to efficient gene delivery must be overcome. One delivery barrier of interest is the enhancement of endosomal escape to prevent vehicle and DNA degradation within the lysosome. However, to properly investigate the generation of analogues designed to enhance endosomal escape, one must also develop a gene delivery vector capable of addressing the deficiencies of traditional cationic polymer vectors.
The overall scope of this thesis project is to address the deficiencies and concerns encountered with traditional non-viral vectors. This has led to the hypothesis involving the development of novel systems based on polyintercalation afforded by incorporation of multiple acridine moieties within a modular polyacridine peptide. Initial studies focused on proof of principle experiments in vitro to assess the polyacridine peptides viability as a gene delivery vector by tethering the fusogenic peptide melittin to polyacridine. Polyacridine-melittin allowed us to conduct SAR (Structure Activity Relationship) studies relating to the sequence and structure of the polyacridine peptides using biophysical measurements and luciferase expression levels in cell culture to dictate peptide design. This data led to the discovery of (Acr-Arg)4-Cys as the optimal in vitro polyacridine-peptide scaffold.
(Acr-Arg)4-Cys was chosen as the lead polyacridine peptide for further development for in vivo mouse studies following PEGylation of the C-terminal cysteine. Polyplexes formulated with the (Acr-Arg)4-PEG peptide demonstrated the ability to produce efficient in vivo gene transfer after delayed hydrodynamic (HD) stimulation. Further in vivo polyacridine peptide SAR studies resulted in identification of (Acr-Lys)6-Cys as a PEGylated analogue that offered superior delivery capability by moderating stimulated gene expression comparable to HD pGl3 after a 1 hr delay between formulation dose and hydrodynamic stimulation.
The properties of (Acr-Lys)6-Cys allowed the in vivo study of multi-component complexes composed of polyacridine PEG, N-glycan targeting ligand, and fusogenic peptide to overcome the delivery barriers, most notably endosomal escape and nuclear localization. Multi-component complexes were formulated with 25 μg of pGL3 and liver gene expression was evaluated by bioluminescence imaging (BLI). Multi-component complexes containing polyacridine-PEG, N-glycan targeting ligand, and/or the charge neutral fusogen PC-4 produced detectable luciferase expression. Alternatively, multi-component complexes formed with the cationic fusogen melittin or anionic fusogen JTS-1 were unable to produce a BLI response, suggesting that multi-component complexes are intolerant of excessive charge. Upon further optimization, polyacridine peptides hold great therapeutic potential due to their modular design and unique nucleic acid binding properties to produce delivery vehicles capable of enabling efficient gene transfer in vivo.