Increased alpha-helicity of a supercharged coiled-coil protein increases siRNA delivery efficiency of protein-lipid hybrid vehicle

Abstract

Gene therapy has the potential to treat various diseases and has recently gained new interest due to the deployment nucleic acid based vaccines for COVID-19. Despite these developments, there still remains a need for further development of gene delivery vehicles to increase their safety and efficacy.. We have recently developed a lipoproteoplex (LPP) consisting of a super-charged coiled-coil protein (CSP) and a cationic liposomal carrier, that has the ability to condense nucleic acids and deliver them in vivo. The LPP is distinct from other liposomal gene delivery systems in that it utilizes a modular protein component to drive transfection activity as opposed to relying on the passive effects of the cationic lipids. A CSP library has been rationally designed to improve the efficacy of the LPP compared to the parent protein via improved alpha-helical structure and increased nucleic acid binding through the use of extended histidine tags and increased positive charge. The secondary structure and nucleic acid binding ability of each library member was assessed, then compared to functional transfection data in NIH-3T3 mouse fibroblasts. Structural and functional data suggests that increasing alpha-helicity of the protein component of the LPP compared to the parent sequence doubles nucleic acid binding affinity and increases transfection activity almost 3-fold with a favorable safety profile.

Supercharged coiled‐coil protein with N‐terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex

“…Expression and purification of the mutant CSP variants were conducted with minimal adjustments to our previously described protocol. 20 Plasmids were transformed via heat shock into chemically competent AF-IQ E. coli cells and incubated on tryptic soy agar plates containing 0.2-μ g/μL ampicillin and 0.034 μg/μL chloramphenicol to grow for 12-16 h at 37 C. Single colonies were then selected following incubation and used to inoculate liquid starter cultures containing 5 mL of lysogeny broth (LB) supplemented with the same antibiotics.…”

“…Transfection complexes were prepared in a previously described two-step process. 16,20 The desired CSP variant was mixed with siGFP…”