Date Published: June 4, 2019
Publisher: Public Library of Science
Author(s): Adam Biddlecome, Habtom H. Habte, Katherine M. McGrath, Sharmila Sambanthamoorthy, Melanie Wurm, Martina M. Sykora, Charles M. Knobler, Ivo C. Lorenz, Marcio Lasaro, Knut Elbers, William M. Gelbart, Sonali Chaturvedi.
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.
The delivery of RNA genes has great potential in a range of therapeutic applications[1–5], with the advantage – compared to DNA gene delivery – that there is no nuclear localization and thus no possibility of genomic integration. Some disadvantages it poses for gene delivery include its vulnerability to ribonuclease (RNase) digestion and – in the case of non-vaccine genes – its triggering of innate immune responses[6,7]. Another limitation for in vivo applications is that gene expression in targeted cells does not have any amplification, resulting in transient and low expression levels. Accordingly, a gene delivery platform that includes self-amplifying mRNA inside a protective capsid allowing for cell targeting and uptake[8–11] could represent a major step forward in mRNA-based gene therapy. We address these issues by using viral replicons (self-replicating RNA molecules) for the self-amplification, and in vitro self-assembled virus-like particles (VLPs) for the protection, specifically using the RNA-dependent RNA polymerase (RdRp) from Nodamura virus (NoV) and capsid protein from Cowpea Chlorotic Mottle virus (CCMV).
We have shown that levels of DC activation are significantly enhanced by incorporation of reporter genes and model antigens into self-replicating RNA molecules – replicons – that have been in vitro packaged into VLPs. This enhancement of marker protein expression has been correlated with a much stronger amplification of RNA replication of the replicons, corresponding to RNA levels as much as ten-thousand-fold greater than those of the housekeeping protein beta-actin. Further enhancement of DC activation and RNA replication – likely facilitated by Fc- receptor-mediated VLP uptake by DCs – has been achieved by pre-incubation of the VLPs with antibodies elicited against capsids of the proteins used to package the replicons. And this amplification is seen in dendritic cells from different donors and for different reporter and vaccine RNA genes. Preliminary in vivo experiments involving vaccination of mice with antigen-replicon VLPs show a small but significant generation of antigen-specific T cells that are doubly positive in their induction of IFN and TFN cytokines.