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1.
Mol Pharm ; 21(4): 1662-1676, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38504417

ABSTRACT

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.


Subject(s)
Mesenchymal Stem Cells , Tissue Engineering , Transfection , Collagen/metabolism , Mesenchymal Stem Cells/metabolism , Lipids
2.
Mol Ther ; 31(2): 374-386, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36303436

ABSTRACT

Emerging and re-emerging viruses, such as Zaire Ebola virus (EBOV), pose a global threat and require immediate countermeasures, including the rapid development of effective vaccines that are easy to manufacture. Synthetic self-amplifying RNAs (saRNAs) attend to these needs, being safe and strong immune stimulators that can be inexpensively produced in large quantities, using cell-free systems and good manufacturing practice. Here, the first goal was to develop and optimize an anti-EBOV saRNA-based vaccine in terms of its antigen composition and route of administration. Vaccinating mice with saRNAs expressing the EBOV glycoprotein (GP) alone or in combination with the nucleoprotein (NP) elicited antigen-specific immune responses. GP-specific antibodies showed neutralizing activity against EBOV. Strong CD4+ T cell response against NP and GP and CD8+ T cell response against NP were detected by ELISpot assays. Intramuscular vaccination with saRNAs conferred better immune response than intradermal. Finally, mice vaccinated in a prime-boost regimen with saRNAs encoding both GP and NP or with GP alone survived an EBOV infection. In addition, a single dose of GP and NP saRNAs was also protective against fatal EBOV infection. Overall, saRNAs expressing viral antigens represent a promising vaccine platform.


Subject(s)
Ebola Vaccines , Ebolavirus , Hemorrhagic Fever, Ebola , Animals , Mice , Hemorrhagic Fever, Ebola/prevention & control , Antibodies, Viral , Antibodies, Neutralizing , Ebolavirus/genetics , Glycoproteins/genetics , Ebola Vaccines/genetics
3.
Mol Ther Nucleic Acids ; 25: 483-493, 2021 Sep 03.
Article in English | MEDLINE | ID: mdl-34589271

ABSTRACT

Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.

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