Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules.

Endosomolysis, a critical barrier to efficient delivery of macromolecules such as nucleic acids, has been breached using a novel approach: endosomolysis by masking of a membrane-active agent (EMMA). To demonstrate the concept of EMMA, a cationic membrane-active peptide, melittin, was reversibly inhibited using a maleic anhydride derivative. At neutral pH, the lysines of melittin are covalently acylated with the anhydride, thereby inhibiting melittin's membrane disruption activity. Under acidic conditions such as those present within endosomes, the amide bond of the maleamate is cleaved, thus unmasking melittin. The active melittin can then disrupt the endosomal membrane resulting in release of biologically active molecules into the cytoplasm. This approach avoids cellular toxicity by restricting melittin's activity until it reaches the endosomal compartment. The utility of this approach was demonstrated by delivery phosphorodiamidate morpholino oligonucleotides (PMOs).