An investigation of sulfhydryl cross-linked DNA delivery systems for liver targeted gene therapy

In order to proceed to any stage of clinical development, a delivery system must be rigorously characterized and meet appropriate specifications, including formulation properties, stability, and biodistribution profiles. Hence, my research has focused on characterizing the formulation properties of nonviral gene delivery systems and correlating formulation parameters with biodistribution and in vivo expression.; First, two PEG-peptides were prepared by conjugating PEG-vinyl sulfone and PEG-orthopyridyl-disulfide with the sulfhydryl of Cys-Trp-Lys₁₈ (CWK₁₈) resulting in the formation of non-reducible (PEG-VS-CWK ₁₈) and reducible (PEG-SS-CWK₁₈) PEG-peptides. They produced small particles (90 nm in mean diameter) with a reduced zeta potential of +10 mV compared to +35 mV determined for non-PEGylated DNA condensates. Also, PEGylation stabilized particles against aggregation upon freeze-drying and rehydration and permitted a higher concentration of particles up to 5 mg/ml. Regarding their biological activity, PEG-VS-CWK₁₈ DNA condensates were more effective in term of blocking in vitro gene transfer than PEG-SS-CWK₁₈ DNA condensates. The results suggest that PEG-peptides can be used as DNA condensing agents that increase DNA condensate solubility and/or can be helpful to extend circulation times of DNA condensates and to enhance access to target cells.; To generate stabilized gene formulations suitable for i.v. dosing, a panel of novel synthetic peptides (dp 20) capable of forming interpeptide disulfide cross-linking when bound to DNA was derivatized with an N-glycan or PEG. Effective targeting to hepatocytes was achieved in mice when condensing DNA with an add-mixture of glycopeptide, PEG-peptide, and a backbone peptide. In addition, it was found that the catabolism of the backbone peptide could influence the liver half-life of the DNA whereas the strength of the cross-linking had no effect on the rate of DNA metabolism. However, an equivalent level of gene expression was obtained for all the optimized formulations containing the ligand for the receptor compared to the negative controls which lack expression. These results illustrate, for the first time, the application of sulfhydryl cross-linked DNA co-condensates for targeted gene delivery to the liver, and that formulation properties are important parameters in determining the in vivo disposition profiles and efficacy of a gene delivery system.