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Neutral Lipopolyplexes for in Vivo Delivery of Conventional And Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine Federico Perche, Rudy Clemençon, Kai Schulze, Thomas Ebensen, Carlos Guzman, Chantal Pichon To cite this version: Federico Perche, Rudy Clemençon, Kai Schulze, Thomas Ebensen, Carlos Guzman, et al.. Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine. Molecular Ther- apy - Nucleic Acids, Nature Publishing Group, 2019, 17, pp.767-775. 10.1016/j.omtn.2019.07.014. hal-02321815 HAL Id: hal-02321815 https://hal.archives-ouvertes.fr/hal-02321815 Submitted on 23 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original Article Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine Federico Perche,1 Rudy Clemençon,1 Kai Schulze,2 Thomas Ebensen,2 Carlos A. Guzmán,2 and Chantal Pichon1 1Centre de Biophysique Moléculaire, UPR4301 CNRS Rue Charles Sadron Orléans, Orléans Cedex 02, France; 2Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany Nucleic acid vaccination relies on injecting DNA or RNA tiveness in controlling tumor growth over mRNA lipoplexes and coding antigen(s) to induce a protective immune response. mRNA polyplexes.15,26 The first generation of mannosylated LPPs RNA vaccination is being increasingly used in preclinical and were formed with the PEGylated histidylated polylysine (PEG- clinical studies. However, few delivery systems have been HpK) cationic polymer and lipophosphoramidate liposomes made reported for in vivo delivery of RNA of different sizes. Using with N-methyl imidazolium lipophosphoramidate (cationic lipid), a tripartite formulation with RNA, cationic polymer, and histamine lipophosphoramidate (protonable lipid), and a mannosy- anionic liposomes, we were able to encapsulate RNA into lated lipid to favor endocytosis by DCs.7 The second generation of neutral lipopolyplexes (LPPs). LPPs were stable in vitro and mannosylated LPPs were also formed with the PEG-HpK polymer successfully delivered conventional RNA and replicative RNA and lipophosphoramidate liposomes but included a glycolipid to dendritic cells in cellulo. Their injection led to reporter containing a tri-antenna of a-D-mannopyranoside instead of a gene expression in mice. Finally, administration of LPP- monovalent mannose for improved endocytosis by DCs.27 Cationic Replicon RNA (RepRNA) led to an adaptive immune response and mannosylated LPPs exerted powerful anti-tumor effects against the antigen coded by the RepRNA. Accordingly, LPPs when used as therapeutic vaccine in different experimental tumor may represent a universal formulation for RNA delivery. models.14,15 INTRODUCTION In addition to complexing conventional mRNA into stable nanopar- RNA vaccination is an expanding field with applications from cancer ticles, a further improvement in RNA vaccination could be gained by immunotherapy, infectious diseases, tissue regeneration and protein the use of self-amplifying RNA or Replicon RNA (RepRNA), partic- – replacement therapy.1 3 Contrary to DNA, RNA does not need ularly for RNA vaccines against influenza.28,29 Auto-amplifying to cross the nuclear envelope for expression, a feature that results RepRNA is generated from a viral genome, a positive-RNA strand in higher transfection efficiency over DNA of differentiated or that bears the ability to replicate and translate without generating in- – non-dividing cells such as neurons4 6 or dendritic cells.7 However, fectious progeny because it lacks at least one structural gene.30,31 nuclease degradation and inefficient or short-time expression of Compared with mRNA, RepRNA allows amplification of RNA copies RNA still limits its application.1,8 While local intranodal injection in the cytoplasm of host cells, which offer several rounds of antigen – of RNA results in effective induction of a specific immune production.32 35 As there are no DNA intermediates, there is no – response,9 11 intramuscular (i.m.), subcutaneous, or intravenous risk of integration. Injection of RepRNA extends the duration (i.v.) injections would be preferred for large-scale preventive vaccina- and magnitude of antigen expression compared to mRNA, giving tion. For that, different types of nanoparticles, from micelles to lipidic equivalent protection against influenza at lower doses than nanoparticle RNA-encoded antigens, need to be presented to lym- mRNA.3,26,36,37 Since RepRNA based-vaccination is a more recent phocytes by dendritic cells (DCs) to induce an immune response strategy than vaccination with mRNA, there are few formulations against cancer or viruses.1,12 Liposome- or lipidic nanoparticle- developed for their delivery.1,3,28 mediated delivery of RNA encoding cancer or viral antigens resulted in protective immune responses in several models (mice and In this study, we evaluated the capability of neutrally charged LPPs, – Macaque) and in humans.1,7,13 23 Among the strategies aimed at including a mannosylated lipid, to transfect DCs.7,14,15 We deter- inducing antigenic presentation by antigen-presenting cells, systemic mined the physico-chemical properties of LPPs and their efficiency delivery of RNA to splenic DCs reached clinical trials.16,24 Kranz 16 et al. used negatively-charged unPEGylated lipoplexes without Received 19 February 2019; accepted 17 July 2019; ligand, which selectively accumulate in the spleen to transfect splenic https://doi.org/10.1016/j.omtn.2019.07.014. DCs and induce a protective immune response against melanoma. Correspondence: Federico Perche, Centre de Biophysique Moléculaire, UPR4301 Our group has developed ternary complexes comprising a cationic CNRS Rue Charles Sadron Orléans, Orléans Cedex 02, France. polymer and mannosylated liposomes, designated lipopolyplexes E-mail: [email protected] 25 Correspondence: Chantal Pichon, Centre de Biophysique Moléculaire, UPR4301 (LPPs), for mRNA delivery to splenic DCs. We previously demon- CNRS Rue Charles Sadron Orléans, Orléans Cedex 02, France. strated that mRNA LPPs show superior immunogenicity and effec- E-mail: [email protected] Molecular Therapy: Nucleic Acids Vol. 17 September 2019 ª 2019 The Author(s). 767 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Molecular Therapy: Nucleic Acids – Figure 1. Formation of LPPs + – – + + + – – + + + – + + + 16 – – et al. on splenic DC transfection with RNA mRNA PEI Polyplexes Liposomes LPP lipoplexes, 40% DOPE helper lipid was included. Liposomes were monodisperse with a diameter of 140–150 nm and a polydispersity to transfect DCs both in vitro and in vivo. Administration of LPPs index (PDI; measure of size distribution) below 0.2 (PDI 0.14/0.05) prepared with conventional RNA or self-replicative RNA resulted and a negative charge of À18/6 mV. Next, we determined the lipo- in reporter gene expression in mice and the induction of an anti- some/PEI ratio yielding neutral LPPs (Table 2). Entrapping the poly- gen-specific immune response when LPPs were prepared with RNA plexes increased the liposome diameter from 140 to 160–190 nm and encoding an influenza antigen. decreased the size distribution, resulting in monodisperse LPPs (PDI 0.13–0.15), features observed previously.14,60,61 Moreover, formation RESULTS AND DISCUSSION of LPPs shielded the positive charges of polyplexes (+20 mV), Formation of Neutral RNA LPPs resulting in neutrally charged LPPs (0.5–2.5 mV), supporting encap- A majority of previous studies reported the functionality upon in vivo sulation of polyplexes in liposomes. Encapsulation was verified by injection of either positively-charged7,15,38 or negatively-charged16,39 electron microscopy (Figures 2B and 2D) and was in accordance RNA complexes. In this study, we chose to use a neutral formulation, with previous studies.60,62 A DOPA/PEI molar ratio of 23 was used as they are known to have benefits over the others. Since, neutral RNA for further studies. Note that complexes prepared with self-replicative lipoplexes are unstable,16 we used an LPP strategy to form neutral RNA, which is larger than concentional mRNA (10,000 nt for VEE- RNA complexes. We produced neutral nanoparticles that would GFP RNA versus 1,000 nt for GFP RNA), were also under 200 nm benefit from their prolonged blood circulation compared to posi- and neutral: 180/10 nm and 1/0.9 mV. – tively-charged and negatively-charged nanoparticles.40 44 Neutral nanoparticles show negligible protein adsorption and decreased com- Systemic administration of RNA complexes requires avoiding aggre- plement activation compared to positive and negative ones.40,42,45 gation or destruction of complexes in physiological fluids and protec- Combined with PEGylation of the liposomes, the neutral surface is tion from RNase degradation.1 Protection against RNase degradation expected to extend blood circulation and increase the chances of was checked by gel electrophoresis (Figure 2A). Whereas naked RNA nanoparticle retention in the desired organs after i.v. injection.42,44,46
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