Enhanced Gene Delivery Mediated by Incorporation of Recombinant Fusion Proteins: Listeriolysin O and Peptides Derived from Protamine.

Abstract

One of the important requirements for non-viral gene delivery systems is to achieve high levels of transfection efficiency by overcoming extracellular and intracellular barriers that exogenous genes need to pass through for expression. The neutralization and condensation of anionic DNA with cationic carriers improve cellular binding and uptake, as well as help protect the DNA from enzymatic degradation in the extracellular environment. Most non-viral vectors are internalized by cells in endocytic compartments and ultimately degraded by hydrolytic enzymes along the endolysosomal pathway. Therefore, promoting endosomal escape of most non-viral vectors confers a significant improvement in transfection efficiency. In order to overcome several important biological barriers, genetically engineered fusion proteins containing multiple functional domains have been recently studied as efficient non-viral gene carriers. In this thesis, we focused on listeriolysin O (LLO) and protamine fusion proteins as one example of recombinant fusion protein-based non-viral multifunctional carriers. The genetically engineered fusion proteins incorporated LLO, the endosomolytic pore-forming protein from Listeria monocytogenes, at the N-terminus and a fragment of the DNA-condensing polypeptide protamine (PN) at the C-terminus. Along with successful expression and purification of LLO-PN and LLO-PNPN fusion proteins, we demonstrated dramatic enhancement of gene delivery efficiency of protamine/DNA complexes with LLO-PN fusion protein and further improvement with LLO-PNPN fusion protein both in vitro and in vivo. Moreover, the association of anionic liposomes composed of PE and CHEMS with LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge to reduce potential interactions with plasma proteins, showed improved in vitro transfection efficiency in the presence of serum and in vivo gene expression in spleens and lungs of mice following intravenous administration. The cleavage of a cathepsin D-cleavable linker introduced between LLO and PN was not observed in vitro, and the linker did not affect the transfection efficiency. The applicability of this delivery system in DNA vaccines was also demonstrated, though limited, generating weak immune responses in mice. Taken together, this study suggests that incorporation of a recombinant LLO-protamine fusion protein in a non-viral vector is a promising strategy that possesses the potential to improve transfection efficiency of non-viral gene delivery systems.