In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA

Abstract

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Keywords: Nodamura replicon; cowpea chlorotic mottle virus (CCMV); mRNA vaccine; plant virus-like particles (VLPs); self-amplifying mRNA; tobacco mosaic virus (TMV).

Figure 1.

(A) Schematic of the mRNA cassettes created by using Biorender. (B) Electron micrographs, median diameters, and lengths of RNaseA-treated CCMV and TMV VLPs containing Nod.LucOAS and Nod.EYFPOAS mRNA cassettes. (C) Electron micrographs and median diameters and lengths of non-RNaseA-treated CCMV and TMV VLPs containing Nod.LucOAS and Nod.EYFPOAS mRNA cassettes. (D) Agarose electrophoresis gels (0.85% in 1 × TAE, pH 8.0) of RNA extracted post RNase treatment and without RNase treatment from Nod.LucOAS and Nod.EYFPOAS mRNA-containing CCMV and TMV VLPs. The lane labeled “mRNA” is the in vitro transcribed RNA used in the assembly reactions. The extent (percentage) of degraded RNA extracted from RNase- and non-RNase-treated VLPs is determined from the density of RNA bands analyzed using ImageJ software.

Figure 2.

Expression of reporter genes upon transfection into BHK cells of 500 ng of mRNA in non-self-amplifying (EYFP and Luc) and self-amplifying/replicon (Nod.EYFPOAS and Nod.LucOAS) form, using lipofectamine seeded into the 6-well cell culture plate (8 × 10⁵ BHK-21 cells/well). (A) Expression of EYFP was captured with fluorescence microscopy; scale bar represents 400 μm. (B) Fluorescent cell population of EYFP levels from mRNA and replicon RNA was determined by fluorescence-activated cell sorting (FACS). The graphs are plotted with side scatter area [FSC-A] versus EYFP fluorescence intensity. For statistical significance, p value was calculated using unpaired two-tailed test. * p < 0.005. (C) Expression of Renilla luciferase for mRNA (Luc, EYFP) and replicon forms of the reporter gene, measured from the cell lysate collected post transfection. Multifold change of Renilla luciferase and EYFP mRNA levels for mRNA and replicon forms in transfected BHK-21 cells was determined by quantitative real-time PCR. The levels of respective mRNAs in the transfected cells were normalized by GAPDH mRNA.

Figure 3.

In vitro delivery of reporter gene replicons in TMV and CCMV VLP-packaged forms, using lipofectamine-mediated transfection into BHK cells. (A) Expression of Renilla luciferase was measured from the cell lysates collected at different times after transfection of naked NodLuc.OAS and of RNaseA-treated (+) and non-RNaseA-treated (−) Nod.lucOAS-containing TMV and CCMV VLPs (750 ng of Nod.LucOAS loaded into the TMV ~ 14 μg and 1000 ng of Nod.LucOAS loaded into the CCMV ~ 3.4 μg). Expression of EYFP was measured at excitation/emission wavelengths of 500 and 540 nm from the transfected cells collected at different times after transfection of naked Nod.EYFPOAS and of RNaseA-treated (+) and non-RNaseA-treated (−) Nod.EYFPOAS-containing TMV and CCMV VLPs (500 ng of Nod.EYFPOAS loaded into the TMV ~ 10 μg and the CCMV ~ 1.7 μg). (B) Multifold changes of luciferase and EYFP mRNA levels with respect to the control cells up to 48 h in the transfected BHK-21 cells were measured by quantitative real-time PCR for naked RNA and for RNase-treated TMV and CCMV VLPs. The levels of respective mRNAs in the transfected cells were normalized by the GAPDH mRNA. For statistical significance, p value was calculated by using two-way ANOVA: ***p < 0.0001, **p < 0.005, *p < 0.05, ns: not significant.

Figure 4.

Bright-field and fluorescence microscopy images of BHK cells transfected with reporter gene replicons in naked and in TMV and CCMV VLP-packaged forms. Expression of EYFP was imaged after transfection of 500 ng of the Nod.EYFPOAS replicon in naked RNA form and in RNaseA-treated (+) and non-RNaseA-treated (−) CCMV and TMV VLPs. The scale bar is 400 μm.

Figure 5.

In vivo delivery of genes using VLPs. (A) Lymph node trafficking and localization of VLPs in the draining lymph node. The fluorescent VLPs, Cy5.TMV and Cy5.CCMV, were delivered into the mice through footpad injection. The localization of VLPs into the draining lymph nodes was quantified from the popliteal lymph node harvested from the mice post injection from 5 to 720 h. For statistical significance, the p values were calculated using one-way ANOVA. ***p < 0.0001, **p < 0.005, *p < 0.05, ns: not significant.

Figure 6.

In vivo delivery of genes using VLPs. In vivo delivery of the self-amplifying enhanced yellow fluorescent protein gene with the Nodamura replicon (5 μg mRNA) encapsulated in non-RNaseA-treated TMV and CCMV VLPs (100 μg) by subcutaneous injection into the footpads of mice. Upon delivery, the expression of EYFP was measured by the fluorescent signal observed in mice using IVIS spectrum optical imaging system. For statistical significance, p values were calculated using one-way ANOVA. ***p < 0.0001, **p < 0.005, *p < 0.05, ns: not significant.