163. Hepatocyte Targeted Endosomal Escape Agents

Abstract

The successful development of potent non-viral DNA and mRNA delivery agents to transfect liver hepatocytes in vivo requires the use of an endosomal escape molecule. Two major strategies have emerged, one that utilizes membrane lytic peptides, and a second that uses proton sponge polymers. Both approaches would require administration of excess endosomal release agent with a smaller dose of targeted polyplex. The present study compared these approaches by conducting 384-well in vitro transfections on primary hepatocytes, as previously described1. An N-glycan targeted melittin was designed to target hepatocytes via the asialoglycoprotein receptor. The synthesis used a purified triantennary N-glycan from bovine fetuin as an potent high affinity asialoglycoprotein receptor targeting ligand that was conjugated by disulfide bound to an N-terminal Cys on melittin, a 26 amino acid membrane lytic peptide. Following targeted entry into hepatocytes via receptor mediated endocytosis, melittin is bioactivated by reduction of the disulfide bond to release the N-glycan, leading to endosomal lysis. RBC hemolysis assays established the membrane lytic activity of triantennary-melittin was muted prior to reduction, which allowed it to be safely i.v. dosed in mice. A second strategy involved the modification of PEI by reductive amination with lactose followed by acetylation, resulting in an asialoglycoprotein receptor targeted proton sponge polymer. The resulting LacAcyl-PEI was purified by RP-HPLC and characterized by NMR. The i.v. dosing of LacAcyl PEI in mice demonstrated that it possessed a much greater safety margin than unmodified PEI. These novel endosomal escape agents are being compared for their ability to mediate in vitro gene transfer of glycan targeted DNA and mRNA in miniaturized primary hepatocyte transfection assays.