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.

Protein‐Engineered Functional Materials

“…Another variant Q (Table ), engineered by truncating the COMPcc s by one heptad to form C (Table ) and swapping regions with respect to glutamine 54, self‐assembles into nanofibers and upon binding to a small molecule, curcumin, forms microfibers (Figure b) . Introduction of positively charged residues on the surface of COMPcc s to form a supercharged protein, CSP endows it the ability to effectively deliver genes (Table ) . CSP exhibits greater helicity compared to the parent protein and can bind with nucleic acids and cationic lipids to form spherical particles termed “lipoproteoplexes.” These assemblies are capable of condensing and delivering nucleic acids (Figure c) .…”

Protein based therapeutic delivery agents: Contemporary developments and challenges

“…Even smaller peptides, such as the class of cell penetrating peptides, while relatively unstructured, have yielded efficacious results in animal models [185, 186]. The hybrid of these approaches encompasses unstructured, structural proteins that are decorated with densely charged sites for complex formation [187–189]. These attributes result in a wide collection of protein-based delivery solutions (Table 2).…”

“…The concept of recruiting the additional charge of particular amino acids into the sequence of a larger scaffold with existing structure and/or function has evolved in the last decade with the notion of supercharging well-structured proteins [187–189, 201, 202]. The predecessor to this work is the supercharged variant of green fluorescent protein (scGFP)[187, 188].…”

“…More recently, a positively charged COMPcc (CSP) has been engineered and developed with cationic lipid formulation FuGENE® HD (FG) (Promega, WI, USA) as lipoproteoplexes for gene delivery [189]. CSP has been replaced with arginine at eight solvent exposed residues and thus carries a net positive charge, enabling it to robustly interact with the cell membrane.…”

“…The growing plethora of protein constructs of controllable physicochemical properties at a nanoscopic and supramolecular scale enables the field with a toolkit of solutions to address the gamut of existing therapeutic (macro) molecules [10, 13, 34, 70, 76, 345]. The particular challenges of bioavailability and pharmacokinetics [3, 4, 17–20] have been shown to be addressed with the widest range of protein-based delivery vehicles, including proteins with defined secondary and tertiary structures like HSA [34, 61, 62, 65, 120], coiled-coils [12, 126, 189], cage proteins [13, 100, 160, 163, 165, 242–244], ferritin [199]/apoferritin [200], and vaults [245, 246], as well as macroscopically amorphous structural proteins such as ELPs [30, 76, 91, 144, 149, 150] and gelatin [9, 31, 86], and rationally designed peptides for self-assembly covering peptide amphiphiles [332, 336], MAX family [333, 342], dock-and-lock (DnL) system [334], and the SHIELD network [335]. The examples presented in this review further demonstrate the applicably of these constructs for both passive and targeted delivery modalities.…”