In our general introduction of "In Vivo Self Amplifying RNA research projects", we emphasized how important it is to mimic the natural use of RNA replicons by a wide range of positive-strand RNA viruses, for purposes of high-level protein expression. We featured the particular case of Nodamura virus, with its two-molecule genome consisting of RNA1 coding for the RNA replicase (RdRp) and RNA2 coding for the capsid protein. One way to use this system for delivery of genes of interest (GOIs) is to simply insert the GOI into the end of RNA1, immediately following a self-cleaving proteolytic sequence, so that the GOI RNA is replicated along with RNA1 and so that its gene product – the desired therapeutic or reporter protein – will be cleaved in functional form from the RdRp. We have done this using EYFP as the reporter gene.
More explicitly, we genetically engineer RNA1 of Nodamura virus to include EYFP as described above, package it in vitro into CCMV VLPs, and then transfect mammalian (baby hamster kidney [BHK]) cells with them. As seen in the figure above, the RNA gene content of the VLP is made available to the translational machinery of the host cell, resulting in reporter gene (here EYFP) expression [Virology 2013].
Presently we are generalizing this scheme to include the synthesis and VLP delivery of several different Nodamura-based RNA replicon systems, containing other GOIs. E.g., in collaboration with our UCLA colleague Prof. Yung-Ya Lin, we are attempting to enhance MRI contrast in cancer cells by delivering replicons to them and over-expressing MRI-contrast-enhancing genes. We are also working with colleagues at Boehringer-Ingelheim to deliver cancer antigen gene in replicon RNA form, for purposes of developing self-amplifying vaccines. For both of these applications it will be important to use the VLP reconstitution methods described earlier, for protecting, targeting, and uptake of the replicons in vivo. We are also working on inserting GOIs that encode non-coding RNA, for up- or down- regulating processes in cells in collaboration with Professor Feng Guo in the Biochemistry department of UCLA.