Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex

Abstract

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

Keywords: Cationic lipid; Coiled-coil protein; Gene delivery; Lipoproteoplexes; Supercharge.

Fig. 1

a) Aligned sequences of COMPcc and CSP with mutated arginine residue positions shown in red. b) Schematic of CSP complexation with plasmid DNA and a ternary complex with cationic lipids to form lipoproteoplexes for gene delivery.

Fig. 2

In vitro studies on CSP and COMPcc. a) CD wavelengths scan of CSP (dashed line) and COMPcc (solid line) at 4 °C at 10 μM concentration. Scans represent an average of three trials. b) Plasmid DNA binding to protein with increasing w/w ratio evaluated through mobility shift assay on 1% agarose gel of CSP binding to DNA (left) and COMPcc binding to DNA (right). In both gels L- 1kb DNA ladder, 1- plasmid DNA alone. Proteoplexes prepared at different protein to DNA w/w ratio: 2 – 0.5:1, 3 – 1:1, 4 – 2:1, 5 – 3:1, 6 – 5:1, 7 – 8:1, 8 – 10:1, 9 – 13:1, 10 – 16:1, 11 – 18:1.

Fig. 3

In vitro transfection efficiency for β-galactosidase DNA. FG•DNA(4:1), COMPcc•DNA (5:1), COMPcc•DNA(5 or 8:1)•FG, CSP•DNA(5:1), CSP•DNA(5 or 8:1)•FG, mTat•DNA(10:1) and mTat•DNA(10:1)•FG. a) * indicates p < 0.0001, comparison of control (DNA only), FG•DNA(4:1) etc, b)* indicates p < 0.0001 comparision of control (DNA only), CSP•DNA(8:1) etc. Data represents the mean β-galactosidase activity (relative light units, RLU)/well with a standard deviation obtained from quadruplicates.

Fig. 4

Cell viability evalauted by MTT assay after treatment of CSP and FG complexed with plasmid DNA and liproteoplex complex with MC3T3-E1 cell. Data is shown as the mean ± standard deviation obtained from quadruplicates and compared to the mean of control group. The control group consist of non-transfected cells (100% viability). No significant difference was observed between CSP•DNA(8:1) and FG•DNA(4:1) or CSP•DNA(8:1)•FG and FG•DNA(4:1) (p < 0.05).

Fig. 5

TEM images of different complexes a) CSP•DNA(8:1), b) FG•DNA(4:1), c) CSP•DNA(8:1)•FG. The scale bars in the image is 500 nm and the insets has 2 μm.