and Abstract This proposal aims to develop targeted nanoparticles to express human factor VIII in liver hepatocytes of mice. A safe and efficient i.v. dosed nanoparticle delivery system to transfect liver hepatocytes to express secreted hFVIII would be transformative for treating hemophilia A. Compared to hydrodynamic dosing of plasmid DNA, all other nanoparticle delivery systems are currently too inefficient to achieve therapeutic levels of protein expression in liver primarily due to the inability of plasmid DNA to traverse the nuclear membrane of non-dividing hepatocytes. The preliminary data establishes that efficient expression can be achieved using double stranded mRNA. Nanoparticles are generated using a novel PEG-peptide containing Lys-Acr residues that binds to ds mRNA. The delivery of ds mRNA nanoparticles to the cytosol circumvents the major barrier that limits expression of plasmid DNA.
The first aim of the proposal will advance ds mRNA nanoparticles by increasing circulatory stability and persistence of expression. Multivalent PEG-peptides will be developed that bind ds RNA with higher affinity to further increase ds mRNA nanoparticles stability following i.v. dosing. Persistent expression will be achieved by developing self-amplifying mRNA constructs designed to replicate mRNA in the cytosol and extend its expression. Efficient hepatocyte targeting will be attained using a high-affinity triantennary N-glycan attached to the PEG-peptide to mediate nanoparticle endocytosis into hepatocytes via the asialoglycoprotein receptor. The potent membrane lytic peptide melittin will be reversible attached to ds mRNA to afford triggered release of mRNA into the cytosol. The optimized ds mRNA nanoparticle delivery system will be used to express human factor VIII with a goal of achieving functional correction of hemophilia A in mice. The successful development of targeted ds mRNA nanoparticles for efficient transfection of liver hepatocytes will advance the field of nanomedicine by establishing a paradigm changing strategy to achieve expression in non-dividing cells following i.v. dosing.