Skip to content Skip to navigation

In Vivo Self-Amplifying RNA Research Projects

                 By “in vivo” experiments we mean ones performed in host cells (rather than in host animals, which is how the term “in vivo” is more generally used, in virology and medical contexts). And by “host cells” we mean controlled monolayers of cells in petri dishes. In this classical form the cells can easily be transfected by RNA or VLPs, or infected by virus, and the transformed cells can easily be assayed in a large number of ways. Ultimately, we would like to transform and assay cells that have been targeted in animals, but we need first to demonstrate and understand how changes of interest can be effected under the controlled conditions of cell culture.


noda lifecycle.png 

Figure 1. The Replication life cycle of the Nodamura Virus. By removing the structural protein gene, RNA 2, a self amplifying RNA can be safely used as a research tool for amplifying genes of interest (GOI).






                  In this context, we are particularly interested in exploiting the unique properties and singular RNA amplification power of “replicon” molecules. A replicon is an RNA molecule that is directly translated by ribosomes and whose gene product is an enzyme that replicates it. (In this sense, it is “self-replicating”.) For example, if you transfect a cell with one copy of a replicon, you will have up to hundreds of thousands of copies of it within hours.      

                 A wide variety of viruses (including Yellow Fever, Dengue, SARS, and Hepatitis C, for example) use this strategy to have their genomes replicated, i.e., their genome is a replicon! And included in the replicon form of their genome are RNA genes coding for viral proteins – other than the RNA replicase – which are also amplified by a factor of thousands.  The figure above illustrates this scenario for the case of the nodamura virus, an insect virus whose genome consists of two RNA molecules (“1” and “2”), both of which are packaged in the same capsid. RNA1 codes for the  RNA dependent-RNA-polymerase (RdRp) – “Protein A” – that binds to RNA1 and to RNA2 and replicates them by first making hundreds of minus (-) strand copies of each and then making hundreds of plus (+) strands from each of the minus strands – see middle, left. In this way up to hundreds of thousands of copies of the genome – pairs of RNA1 and RNA2 – are synthesized, ready to be packaged by capsid protein (a) thereby forming a new generation of infectious virus.

                Most significantly for our interests,  RNA2 can be replaced by an RNA molecule encoding any gene of interest to us (or, for that matter, non-coding RNA). The new RNA simply has to contain the 5’ and 3’ sequences needed for it to be bound and replicated by the RdRp synthesized from RNA1. That is, by simply removing the capsid protein gene of RNA2 and replacing it with a designed RNA sequence, we can strongly amplify – in situ, i.e., in a cell of interest – any RNA of our choosing.  Further, because we are aiming to do this in the context of whole organisms, we first package the replicon into a VLP, using our in vitro CCMV self-assembly protocols, and then functionalize the VLP to protect it from the immune system and assure its targeting and uptake by specific cells. In this sense, each of the many examples we discuss below involve a mix of in vitro reconstitutions involving purified components (RNA, capsid protein, and lipid) and tissue-culture (“in vivo”)  transfection and analyses of host cells.