David L Lewis — Page 2

PubMedDate not supplied

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).

Cytoplasm↗

PubMedDate not supplied

Hepatocyte targeting of nucleic acid complexes and liposomes by a T7 phage p17 peptide.

A critical step for liver-directed gene therapy is the selective targeting of nucleic acids to hepatocytes. We have previously discovered that the proximal half of the T7 phage tail fiber protein (p17) targeted intact T7 phage and recombinant proteins to hepatocytes in vivo. In the present study, we have localized the targeting activities to a 33 amino acid sequence within the p17 coiled-coil rod domain. Given that the tail fiber domain from which the peptide was derived may form alpha and triple helical structures, biophysical studies (CD spectra and analytical ultracentrifugation) were conducted to determine the secondary and tertiary structures of the peptide. This peptide is able to target proteins, polymers, and siRNA and also particles such as DNA polyplexes and liposomes to hepatocytes. A variety of coupling strategies and chemistries were employed, thus demonstrating that this peptide is a versatile system for delivering cargo. The ability of this hepatocyte-targeting peptide to target DNA-containing particles suggests that it should be useful in the development of both nonviral and viral vectors. However, biological function of delivered cargo has not been demonstrated. This was primarily due to failure of delivered cargo to escape the endosomes. Further studies are in progress to provide functional activity of delivered nucleic acids by enabling their endosomal escape.

Amino Acid Sequence↗