UCLA

We have genetically engineered the capsid proteins (CPs) of two RNA-packaging plant viruses, BMV and TYMV, to facilitate the presentation of arbitrary protein ligands on the exterior surface of the virus-like particles (VLPs) that they form. This is important for the targeting of in vitro self-assembled mRNA-containing VLPs for in vivo gene delivery purposes. In particular, this platform will provide an alternative to lipid nanoparticle systems, with the advantages: of ensuring definite stoichiometry – one mRNA molecule per 180-protein capsid (as opposed to an indeterminate number of mRNAs in polymorphic/mixed-component liposomes); and of precise control over the site of conjugation of protein ligands, introducing genetic fusions (as opposed to promiscuous N-Hydroxysuccinimide (NHS) esters or sulfhydryl-maleimide chemistry).More explicitly, we have developed two novel approaches to the functionalization of in vitro reconstituted VLPs with protein ligands. The first involves the genetic insertion of a poly-Glycine (polyG) moiety into an exterior loop of a CP, just downstream of a protease cut site. Cutting of the loop, followed by the addition of the Sortase A enzyme, will result in covalently linking – to the newly created polyG N-terminus – an arbitrary protein to whose C-terminus contains the Sortase A binding motif (LPXTG). The second strategy involves exploiting an analogy between the beta barrels of viral CPs and of green fluorescent protein (GFP) “split proteins.” Here the idea is to make the split portions of CPs, genetically fuse the N-terminus of the C-terminal part (i.e., the tail end of the CP) to a protein ligand of interest, and then mix the two CP portions together so that they associate into a nicked mutant CP that, in turn, self-assembles into VLPs with protein ligands already displayed on their exterior surfaces. These two strategies are demonstrated for BMV and TYMV, respectively, and offer “proof-of-principle” examples of a potentially powerful means for in vitro synthesis of targeted mRNA-containing particles for gene delivery.