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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 chloroticmottle 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.
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This content will become publicly available on August 1, 2026
A New Approach to In Vivo Transformation of Killer T Cells
Chimeric antigen receptor (CAR) T cell therapy is a relatively new and powerful way of transforming T cells with receptors needed to recognize and kill diseased cells. Traditionally, it involves extraction of T cells from a patient, ex vivo transformation of them with CARs, expansion, and subsequent re-infusion into the patient. Recent developments aim to avoid this lengthy, costly patient-specific procedure by using var- ious viral and non-viral vector particles for direct in vivo delivery of CAR-encoding genes. In this paper we highlight several fundamental connections between in vitro and in vivo aspects of this process. We dis- cuss the proposed use of in vitro-reconstituted virus-like particles (VLPs), prepared from purified CAR- encoding mRNA and viral capsid protein, and functionalized with a T cell-targeting antibody. We compare and contrast these particles – and their use as gene vectors – with the several modalities currently employed that involve in cellulo generation of lentiviral or AAV vectors or in vitro complexation of nucleic acids with cationic polymers or lipid vesicles. We report the unique stoichiometric preciseness and ther- modynamic stability of VLPs formed from anti-HIV-glycoprotein CAR-encoding mRNA and the capsid pro- tein from a plant virus, and quantify the extent to which these monodisperse spherical VLPs are RNase resistant and lead to strong CAR expression in T cells. Further, in vitro cell-killing experiments are pro- posed, in which these CAR VLP-transformed T cells are mixed with HIV-infected cells, to be followed by in vivo experiments involving injection of the particles into HIV-infected humanized mice.
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- Award ID(s):
- 2413062
- PAR ID:
- 10631861
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Molecular Biology
- ISSN:
- 0022-2836
- Page Range / eLocation ID:
- 169369
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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