Abstract
Non‐viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge‐based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein‐protein and protein‐DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia‐related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM‐based DNA scaffolds combine interesting characteristics in an easy‐to‐use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein‐based gene delivery.
Highlights
Despite the progress in gene therapy research and the availability of a few gene therapy products, the safe and efficient delivery of DNA remains challenging
In the presence of plasmid DNA, the phospholipase C (PLC)-TFAM protein retained 50% of its PLC activity compared to the free protein (Figure S1)
When cysTFAM-vaccinia-related kinase 1 (VRK1) was replaced with cysTFAM-NLS, containing a c-terminal nuclear localization signal, the transfection efficiency of the system decreased to almost basal levels
Summary
Despite the progress in gene therapy research and the availability of a few gene therapy products, the safe and efficient delivery of DNA remains challenging. The most commonly used nucleic acid delivery systems are viruses and synthetic particles made from various materials such as lipids or polymers.[1] While viruses are highly efficient delivery vehicles, they pose several threats, have limited packaging capacity and cannot be modified without losing efficacy. The assembly of non-viral systems, on the other hand, is often based solely on electrostatic or hydrophobic interactions (e.g., ionizable lipids or dendrimers). In the presence of serum proteins, such particles are prone to destabilization and aggregation. These systems interact poorly with the cellular machinery, for example specific cell surface receptors or the cellular transport machineries. This usually translates into low transfection efficiencies.[2]
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