Targeted Delivery of Self-Amplifying RNA Genes

We design and synthesize self-replicating forms of messenger RNA coding for reporter and therapeutic genes, to be packaged into CCMV or BMV virus-like particles by in vitro reconstitution. The basic idea is to use the RNA-dependent RNA polymerase (RdRp) of a positive-sense/ready-to-translate viral genome. The 1^st molecule (RNA1, ~3000nt) of the RNA genome of Nodamura virus, for example, is directly translated to give an RdRp that strongly replicates it. By adding a gene of interest to RNA1, that gene – coding for a reporter like EYFP or luciferase, or a therapeutic protein like a viral or cancer antigen – is strongly replicated along with the RdRp gene and translated to give the target protein of interest. The resulting construct – a single-molecule replicon – is still short enough (~4000nt) to be in vitro reconstituted in CCMV or BMV VLPs, which can then be functionalized by targeting ligand or wrapped in lipid bilayer and used for gene delivery.

MicroRNAs have been actively studied for their potential application to treatment of a wide variety of diseases – both in the context of gene regulation at the translational level, and of transcriptional silencing and direct interaction with regulatory proteins. Using RNA replicase genes from positive-sense RNA viruses, we have constructed self-replicating forms of microRNAs in analogy with amplification of mRNAs in our gene and vaccine delivery work. The miRNA sequence of interest is flanked by self-cleaving ribozymes and added to the replicase gene of the Nodamura virus, which is directly translated to generate an RNA-dependent RNA polymerase that replicates it strongly along with the doubly-self-cleaving ribozyme cassette containing the microRNA. Transfection of mammalian cells with replicons of this kind results in the generation of as many as a million microRNAs per cell. In the case of microRNA-34a the biological activity of microRNA amplified in this way is demonstrated by the essentially complete apoptosis of cultured prostate cancer cells transfected with the replicon.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. We developed a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying (“replicon”) mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers – CD80, CD86 and MHC-II – and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction [1].

We are making in vitro self-assembled virus-like particles (VLPs) made from ssRNA and the capsid protein from tobacco mosaic virus (TMV) instead of from CCMV. TMV was the very first virus to be reconstituted from its purified components, and – as with CCMV – its capsid protein is capable of packaging heterologous RNA as long as the TMV RNA “packaging signal” is inserted into the foreign RNA to ensure the nucleation of a capsid. The capsid that forms is a long hollow/helical cylinder of protein instead of an icosahedral spherical shell. Because the curvature (reciprocal radius) of the cylinder remains constant independent of its length, there is no limit to the length of heterologous RNA than can be packaged in this way. This fact allows us – in contrast to the situation with CCMV or BMV VLPs – to accommodate replicons of any length, and to therefore use RdRp genes from mammalian viruses like Sindbis instead of being restricted to RdRps from insect viruses like Nodamura that happen to work – but, not surprisingly, not as well as those from mammalian viruses – in mammalian cells. Further, the packaged RNA is accommodated inside the 7nm-thick protein shell itself, leaving the 4nm-diameter hollow interior available for loading of other therapeutics. Specifically, in collaboration with the Steinmetz group at UCSD we are preparing and packaging RNA replicons derived from the ~7600nt-long RdRp genes of Sindbis, to which we add genes of interest along with the TMV packaging signal; the hollow interior is then loaded with a chemoagent like cis-platin, whose positive charge is opposite to that of the inside surface of the capsid.

We are developing an RNA-based therapy for Yellow Fever Virus (YFV) via the construction of defective-interfering (DI) RNA molecules. The DI RNAs in this case involve specifically engineered deletion mutants of the yellow fever genome that are noninfectious yet still replication-competent; they are replicated by the viral RdRP to the detriment of the genome itself, thereby lowering the viral load. While many viruses, e.g., influenza, have naturally-occurring DIs that allow for a lower-grade/more-persistent infection, YFV does not, so we need to identify one by rational design in the lab.

We are designing, expressing, and purifying recombinant proteins that are fusions of CCMV or BMV capsid protein (CP) with antibodies and other targeting ligands, for purposes of enhancing the targeting specificity of VLP self-amplifying-gene delivery systems. Aside from the usual challenges of assuring strong bacterial expression in soluble fractions, the fusion inserts must be done in a way that doesn’t interfere with the in vitro reconstitution/self-assembly of ssRNA-VLPs. In parallel, we are working on a set of alternative approaches that involve making wildtype-CP VLPs and then functionalizing them with protein ligands using a variety of conjugation- and click- chemistry techniques. Additionally, we are preparing CPs with sortase insertions for facilitating the presentation of arbitrary targeting proteins on the outside surfaces of VLPs.