Engineered MTD4 domain efficiently delivers diverse peptides and proteins intracellularly in vitro and in vivo

The therapeutic potential of many biomacromolecular drugs, including antibodies and peptides, is severely limited by their inability to cross cell membranes and reach intracellular targets. Current delivery methods often lack efficiency, metabolic stability, or broad applicability, creating a significant bottleneck in developing treatments for a vast range of diseases. This challenge leaves many crucial intracellular pathways and proteins considered "undruggable," hindering the development of novel therapies for conditions requiring precise intracellular modulation.

Researchers engineered a family of small (~90 amino acids), metabolically stable membrane translocation domains (MTDs) by modifying the loop sequences of a human fibronectin type III (FN3) domain. The most potent variant, MTD4, was then recombinantly fused to the N- or C-terminus of various functional peptides and proteins. They evaluated the intracellular delivery efficiency of these MTD4 fusions in eukaryotic cells in vitro and assessed their biodistribution and tissue penetration following systemic administration in mice in vivo. The primary endpoint was the successful and efficient translocation of cargo into the cytosol and nucleus.

The engineered MTD4 variant proved to be highly cell-permeable, effectively serving as a versatile delivery vehicle. Fusions with MTD4 efficiently delivered a wide variety of functional peptides and proteins into both the cytosol and nucleus of eukaryotic cells, demonstrating success in vitro. Following systemic administration in mice, an MTD4 fusion protein exhibited broad biodistribution and homogeneous tissue penetration across various organs. Importantly, MTD4 is effective at low nanomolar (nM) concentrations, highlighting its potency. This robust intracellular delivery capability addresses a critical barrier in drug development. > MTD4 fusions efficiently delivered diverse peptides and proteins into the cytosol and nucleus of eukaryotic cells, both in vitro and in vivo, at low nanomolar concentrations.