The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

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.