In Vitro-Reconstituted Virus-like-particle Delivery of Self-Amplifying RNA Genes

The delivery of RNA genes has great potential in a range of therapeutic applications. A couple of main limitations for RNA-based therapies include its vulnerability to ribonuclease (RNase) digestion, and its transient and low expression levels due to its lack of amplification. Accordingly, a gene delivery platform that includes self-amplifying mRNA inside a protective capsid allowing for cell targeting and uptake could represent a major step forward in mRNA-based gene therapy. The self-amplifying nature of viral-encoded RNA-dependent RNA polymerases (RdRp) of Nodamura virus holds promise in the development of a platform for self-amplifying RNA gene delivery. To protect and assist in uptake of mRNA encoding an RdRp coupled to a gene of interest, plant virus-like-particles (VLPs) are a strong candidate. Cowpea chlorotic mottle virus (CCMV) capsid protein has been shown to be able to package in vitro heterologous mRNAs, and also to make the genes available to mammalian cells. To better characterize the uptake and amplification in mammalian cells of RdRp-associated genes (also called a replicon, because of its self-amplifying nature), both when naked or encapsidated, reporter genes such as enhanced yellow fluorescent protein (EYFP) or luciferase were used.In collaboration with the German pharmaceutical company Boehringer Ingelheim (B-I), evaluation of possible efficacy of this platform for a cancer vaccine was explored by incorporation of a model antigen (Ovalbumin epitope, SIINFEKL) sequence into the RNA1 of Nodamura virus. From in vitro studies involving incubation of OVA-replicon-VLPs, we observed that CCMV VLPs can be taken up by dendritic cells, where replication then occurs to promote increased production of antigen. Flow cytometry analysis revealed increased expression of maturation markers, and assaying the dendritic cell content by qPCR showed a significant number of SIINFEKL epitope-containing RNA. After these OVA-replicon-VLPs are injected into mice, a population of SIINFEKL-specific CD8+ (cytotoxic) T cells results

Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

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. In this paper we report 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