Endosomolysis by Masking of a Membrane-Active Agent (EMMA) for Cytoplasmic Release of Macromolecules

Abstract

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).

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

“…7]. These results are consistent with previous studies of irreversible modifications of amphipathic polycations (31) and highlight the necessity for the use of reversible modifications to achieve endosomolysis.…”

“…In our strategy, amine groups on the endosomolytic agent are modified with a maleic anhydride, creating acid-labile maleamate bonds (32). These bonds are cleaved within the acidic environment of the endosome, unmasking the agent's amines and activating its endosomolytic capabilities (31). The endosomolytic agent used in the present study is an amphipathic poly(vinyl ether) we previously developed termed PBAVE, which is composed of butyl and amino vinyl ethers (33).…”

“…The siRNAs must then escape from endosomes to elicit RNAi. To accomplish efficient endosomal escape, we developed a strategy that relies on the selective activation of a latent endosomolytic agent in the acidic environment of the endosome (31). Selective activation ensures that deleterious interactions with other membranes the agent encounters before endocytosis are prevented.…”

Breathing Life into Polycations: Functionalization with pH-Responsive Endosomolytic Peptides and Polyethylene Glycol Enables siRNA Delivery

“…After endosomal acidification the DMMAn protecting groups are cleaved and lytic activity is restored. 7,8 This principle enables the nucleic acid carrier to adapt its behavior to the different needs of extra-and intracellular delivery steps.…”

HIF2α-Targeted RNAi Therapeutic Inhibits Clear Cell Renal Cell Carcinoma

“…The protease cleavable masking reagents were developed to provide the DPC with longer systemic circulation time compared to the pH-labile maleic anhydride (CDM) masking chemistry used in first-generation DPC (29,37). For targeting tissue outside of the liver, longer circulation times are necessary for targeting receptors that are likely less abundant and potentially less efficient than the Ashwell-ASGPr expressed on hepatocytes (30,38).…”