Anne-Line Coolen*, Céline Lacroix*, Laetitia Vachez, Céline Coiffier, Perrine Mercier-Gouy, Gwenaëlle Humbert, Charlotte Primard, Emilie Delaune, Jean-Yves Exposito, Bernard Verrier
Laboratoire de Biologie Tissulaire et d'Ingénierie Thérapeutique, UMR 5305, Université Lyon 1, CNRS, IBCP – Lyon, France
* These authors contributed equally to this work. Correspondence: bernard.verrier@ibcp.fr
Abstract
Messenger RNA-based platform currently raises a growing interest in vaccinology but several challenges remain to overcome. Among them, efficient delivery of antigen-coding mRNA into the cytosol of immune cells is needed to induce antigen production and to trigger a broad spectrum-immune response. Actually, major delivery systems for mRNAs were based on lipid nanoparticles. In this study, we developed an alternative strategy to lipid formulations using poly(lactic acid) nanoparticles (PLA-NPs) as carrier. Because mRNA and PLA-NPs are both negatively charged, their association require the addition of cationic intermediates. To that end, we compared poly-L-lysine, dendrigraft of poly-L-lysine, protamine and three cell-penetrating peptides (RALA, LAH4 and LAH4-L1) as cationic intermediates. We designed and optimized strategies to obtain stable and homogeneous colloidal nanocomplex formulations. For each strategy, the functionality of mRNA was verified on 1-cell stage zebrafish eggs and each transfection efficiency was evaluated in vitro in cell models. Ribonucleic acid platforms based on (1) the formation of precomplexes by condensing mRNA with LAH4 and LAH4-L1 and (2) adsorption of those precomplexes onto PLA-NPs seem to be the best promising. In fact, those formulations are stable, present a polydispersity index close to 0.1 and have the capacity to transfect cells and to influence mRNA expression. Thus, LAH4- and LAH4-L1-based formulations represent a promising platform to vectorize mRNA for vaccine development.
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1. Introduction
Vaccination is one of the major successes in medicine and has been essential for successfully fighting against a large number of diseases2. Although generally very efficient, traditional vaccines based on attenuated or inactivated pathogens present a low infectious risk, which makes them not applicable for a number of infectious pathogens341. To reduce the infection risk, sub-unit and recombinant protein vaccines have been developed. However, to circumvent the low immunogenicity of these vaccines, adjuvants are needed, such as immunostimulatory molecules, or new vectorization systems to enhance their efficiency.
mRNA-based delivery approaches have emerged as highly promising in vaccinology and their development currently receives much attention23. These strategies aim at introducing effectively stable in vitro transcribed (IVT) mRNAs encoding antigenic proteins into the cytosol of target cells, and to induce an adequate immune response. mRNA-based medicament presents key benefits for vaccine development. First, and contrary to proteins, proof-of-concept of a mRNA formulation can be easily adapted for the fast development of a variety of new vaccines. Second, compared to DNA, mRNAs do not integrate into the host genome, minimizing the potential risks of insertional mutagenesis. Third, mRNAs do not need to cross the nuclear envelop for their expression, increasing their ability to transfect non-diving and hard-to-transfect cells such as cells relevant for the immune response. Finally, IVT mRNA chemical modifications and the development of sequences increasing mRNA stability have potentiated their vaccine efficiency35.
Targeting dendritic cells (DCs) is often the goal of mRNA vaccine strategies, as these major antigen presenting cells (APCs) play central roles in inducing and orchestrating immune responses
244,342. Hence, DCs might translate mRNA into antigens, process these proteins for presentation on
the Major Histocompatibility Complex type I (MHC-I) and/or II (MHC-II), and activate the cytotoxic and humoral arms of the adaptive immune response3. The potential for inducing a cytotoxic response is indeed a great advantage of such approaches, for fighting against diseases such as cancer or HIV23,55,118,343. In addition, DCs express PRRs (Pattern Recognition Receptors) such as, innate immunity receptors able to recognize RNAs in the cytosol or endosomes and activate the DCs for initiation of the immune response4,40. Thus, mRNA present a self-adjuvant effect, that, if balanced, can contribute to activate DCs157. Altogether, these advantages make mRNAs highly promising biomolecules for new vaccine therapies or protections.
Despite advantages, key challenge associated with the use of mRNAs is their delivery to DCs. In fact, mRNAs are polyanionic molecules, which in naked format has great difficulty to cross nonpolar cellular barriers to reach the cytosol compartment. Moreover, mRNAs are susceptible to
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rapid degradation by nucleases and must be protected during the delivery. Although important advances have been made to improve the stability and functionality of mRNAs169,344, a major hurdle for using mRNAs remains to identify efficient delivery systems. Various delivery complexes originally developed for DNA and siRNA have been used for mRNA transfection. According to their chemical composition and organization, these vectors can protect mRNA from degradation, favor their cellular uptake and/or endosomal escape182. Actually, lipid nanoparticles (LNP) are the most attractive platform for mRNA delivery24,32,165–167. In fact, although very encouraging results have been obtained with them in preclinical and clinical studies82,166,193, the need to optimize their delivery in DCs remains high in the vaccine field.
Our group has developed a series of vaccine vectors based on nanoparticles (NP) of poly(lactic acid) (PLA), a biodegradable and biocompatible polymer. PLA-NPs have the ability to co-vectorize antigens and/or immunostimulatory molecules, which are either encapsulated or adsorbed, depending on their chemical properties186,255,260,345. PLA-NPs have shown promising results to induce immune responses against several model antigens after parenteral administration in mice and non-human primates186,260,319. Importantly, PLA-NPs are efficiently taken up by DC in vitro and in vivo256,260,263.
Thus, PLA-NPs constitute promising platforms for in vivo delivery of mRNAs to DCs.
In this study, we have tested various methods to vectorize mRNAs into PLA-NPs. PLA-NPs and mRNAs being negatively charged, we have evaluated the capacity of several cationic molecules (dendritic poly(L-lysines) (DGL), poly-L-lysine, protamine and the cell penetrating peptides (CPPs) RALA, LAH4 and LAH4-L1) to adsorb mRNAs onto PLA-NPs, those positively charged intermediates having an efficient capacity to transfect DNA or siRNA336,337,339. For the most promising formulations, we have tested their functionality and their efficiency either by in vitro transfection of DCs or by cytosolic injection in zebrafish eggs. Among them, the most promising strategy consists of a two steps process, with first the formation of precomplexes by condensing mRNA with LAH4 or LAH4-L1, and then the adsorption of those precomplexes onto PLA-NPs. Those formulations are stable and homogeneous and present a great ability to transfect cells and to promote mRNA translation, and are a promising tool in the fields of vaccine development.
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