Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

Abstract

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

Dendritic Cell Targets for Self-Replicating RNA Vaccines

“…This figure shows the non-cytopathic Pestivirus genome, and two forms of replicon that have been generated [2,45,46]. These are replicons lacking a single structural glycoprotein, and replicons lacking all structural glycoproteins.…”

“…Indeed, application of polysaccharide, lipid and lipoplex technologies is showing high potential for replicon RNA delivery [2,40,45,49,50]; cylindrical BSAbased particle delivery have also been reported [51]. The success of synthetic, biodegradable chitosan-based nanogels for protein and DNA delivery to DCs [52,53] was elaborated to display an efficient capacity for transporting replicon RNA into DCs, leading to RNA translation in vitro and immune responses induction in vivo [2].…”

“…Importantly, the replicon RNA was shown to associate physically with these delivery vehicles, confirming the role of the nanoparticulate systems in the actual delivery of the RNA to the DCs. This was important, because the replicon RNA -derived from the non-cytopathogenic Pestivirus classical swine fever virus -was non-infectious, incapable of entering the DCs or inducing an immune response in vivo without the assistance of the delivery vehicle [2,45]. Figure 2: Schematic representation of the non-cytopathic pestivirus, classical swine fever virus (CSFV), genome.…”

“…Indeed, this replicon RNA was co-formulated with pre-formed squalene-based nanoemulsions [54], employing the squalene-based MF59 adjuvant [55]. Nonetheless, cell delivery of replicon RNA by nanoparticulate delivery vehicles [40] has clearly been demonstrated [2,45,51], leading to translation and replication in DCs and the induction of immune responses in vivo.…”

“…Such difficulties do not arise with RNA vaccines, but these suffer from high lability, especially in RNase-containing extracellular and intracellular environments. Delivery of DNA vaccines has provided the means of facilitating RNA vaccine applicability, namely through the employment of biodegradable, nanoparticulate delivery vehicles [2,8,[17][18][19][20][21]. The delivery vehicle can protect the RNA as well as enhancing its delivery to cells, for which interaction with DCs would prove a major contribution to development of immune defences.…”