title: Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.
abstract: To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.
authors.affiliation: Department of Pharmaceutical Sciences, Center for Molecular Drug Targeting, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Na Hyung
authors.initials: NH
authors.lastname: Kim
authors.forename: Chester
authors.initials: C
authors.lastname: Provoda
authors.forename: Kyung-Dall
authors.initials: KD
authors.lastname: Lee
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry*; D018014:Gene Transfer Techniques; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D011479:Protamines / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011479:Protamines; D011993:Recombinant Fusion Proteins; C038705:hlyA protein, Listeria monocytogenes
keywords: fusion protein; listeriolysin O; nonviral DNA delivery; protamine