nanomaterials Review Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives Itziar Gómez-Aguado , Julen Rodríguez-Castejón ,Mónica Vicente-Pascual , Alicia Rodríguez-Gascón , María Ángeles Solinís * and Ana del Pozo-Rodríguez * Pharmacokinetics, Nanotechnology & Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain; [email protected] (I.G.-A.); [email protected] (J.R.-C.); [email protected] (M.V.-P.); [email protected] (A.R.-G.) * Correspondence: [email protected] (M.Á.S.); [email protected] (A.d.P.-R.); Tel.: +34-945013469 (M.Á.S.); +34-945014498 (A.d.P.-R.) Received: 31 January 2020; Accepted: 16 February 2020; Published: 20 February 2020 Abstract: The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted. Keywords: in vitro transcribed messenger RNA (IVT mRNA); gene therapy; nanomedicine; immunotherapy; infectious disease vaccines; cancer immunotherapy; Chimeric Antigen Receptor (CAR) T cells; dendritic cells; protein replacement; gene editing 1. Introduction According to the European Medicines Agency (EMA), a gene therapy medicinal product generally consists of a vector or delivery formulation/system containing a genetic construct engineered to express a specific transgene (‘therapeutic sequence’) for the regulation, repair, replacement, addition or deletion of a genetic sequence [1]. Nevertheless, a broader perspective is usually accepted from a scientific point of view, and the concept of gene therapy includes the therapeutic application of products containing any nucleic acid. Gene therapy entered clinical trials in the early 1990s. Up to date around 17 nucleic acid-based products have been approved worldwide, and almost 2700 gene therapy-based clinical trials have been completed, are ongoing or have been approved for a broad range of applications. It is expected that nucleic acid-based products will have a substantial impact on the biopharmaceutical market in the near future [2]. Gene therapy has been clinically implemented primarily through two different approaches: Ex vivo or in vivo. In ex vivo therapy, cells are harvested from the patient or a donor, in vitro modified with the therapeutic nucleic acid and finally, re-infused into the patient. In vivo gene therapy is applied by direct administration of the vector containing the nucleic acid into the patient [3]. Nanomaterials 2020, 10, 364; doi:10.3390/nano10020364 www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 364 2 of 43 therapyNanomaterials is applied2020, 10, 364by direct administration of the vector containing the nucleic acid into the patient2 of 42 [3]. Depending on the final objective, gene therapy can be applied for gene augmentation, gene Depending on the final objective, gene therapy can be applied for gene augmentation, gene silencing, or gene editing [4]. Diverse nucleic acids are being used to address the development of silencing, or gene editing [4]. Diverse nucleic acids are being used to address the development of these these new medicinal products. DNA and messenger RNA (mRNA) induce protein expression, new medicinal products. DNA and messenger RNA (mRNA) induce protein expression, whereas, whereas, small interfering RNA (siRNA), microRNA, oligonucleotides or aptamers provide gene small interfering RNA (siRNA), microRNA, oligonucleotides or aptamers provide gene silencing [2]. silencing [2]. Molecular scissor and gene editing approaches, such as zinc finger nucleases (ZFNs), Molecular scissor and gene editing approaches, such as zinc finger nucleases (ZFNs), transcription transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR/Cas9) are also being developed. repeats (CRISPR)-associated nuclease Cas9 (CRISPR/Cas9) are also being developed. The specific features of synthetic mRNA make it a promising alternative to DNA based products. The specific features of synthetic mRNA make it a promising alternative to DNA based products. Firstly, mRNA does not need the machinery of the nucleus to be functional, as DNA therapies do [5– Firstly, mRNA does not need the machinery of the nucleus to be functional, as DNA therapies do [5–8]. 8]. Therefore, once mRNA reaches the cytoplasm, translation of the encoding protein begins, being Therefore, once mRNA reaches the cytoplasm, translation of the encoding protein begins, being effective in both, mitotic and non-mitotic cells [9–11]. Secondly, mRNA has a better safety profile, effective in both, mitotic and non-mitotic cells [9–11]. Secondly, mRNA has a better safety profile, because it does not integrate into the host genome; hence, the risk of carcinogenesis and mutagenesis because it does not integrate into the host genome; hence, the risk of carcinogenesis and mutagenesis usually associated with DNA is notably reduced [5–8,11]. In addition, it works without encoding usually associated with DNA is notably reduced [5–8,11]. In addition, it works without encoding additional genes, (i.e., those related to antibiotic resistance) [9]. Thirdly, the synthesis of the encoded proteinadditional is fast, genes, and (i.e.,its expression those related is temporary to antibiotic [6,9]. resistance) The onset [ 9of]. expression Thirdly, the is synthesis usually detected of the encoded within 1hprotein after istransfection fast, and its with expression a peak is in temporary expression [6 ,95–7]. The h later onset [12]. of expression Finally, the is usuallyproduction detected of in within vitro transcribed1h after transfection mRNA (IVT with mRNA) a peak is easier in expression than the 5–7manufacturing h later [12]. of Finally, DNA, and the it production can be standardized, of in vitro maintainingtranscribed mRNAits reproducibility (IVT mRNA) [7]. is easier than the manufacturing of DNA, and it can be standardized, maintainingNevertheless, its reproducibility the use of IVT [7 mRNA]. for clinical purposes has been mostly limited by its physical instability,Nevertheless, its immunogenic the use of capacity, IVT mRNA and for the clinical difficulty purposes in passing has been through mostly the limited cellular by membrane, its physical dueinstability, to the itsanionic immunogenic nature of capacity, mRNA andmolecules the diffi culty[8,13,14]. in passing The knowledge through the of cellular mRNA membrane, structure has due boostedto the anionic modifications nature of mRNAdesigned molecules to improve [8,13 ,14stability]. The knowledgeand translation of mRNA efficacy, structure and hasto boostedreduce immunogenicity;modifications designed however, to improve optimized stability IVT mRNA and translation still has etoffi cacy,overcome and to several reduce extracellular immunogenicity; and intracellularhowever, optimized barriers. IVT As mRNA observed still hasin Figure to overcome 1, IVT several mRNA extracellular has to avoid and intracellular degradation barriers. from extracellularAs observed degradative in Figure1, IVT agents, mRNA such has as to ribonuclea avoid degradationses, and interact from extracellular with the target degradative cell, cross agents, the cytoplasmicsuch as ribonucleases, membrane and and interact diffuse with in the the cytoplas target cell,m to cross reach the the cytoplasmic ribosomes membrane [7,15]. The and formulation diffuse in ofthe IVT cytoplasm mRNA in to suitable reach the nanosystems ribosomes [or7,15 vectors]. The is formulation frequently ofa requirement IVT mRNA infor suitable surpassing nanosystems all these barriers.or vectors Non-viral is frequently vectors, a requirement and more for specifical surpassingly lipidic all these systems, barriers. are Non-viral the most vectors, studied and among more deliveryspecifically systems lipidic for systems, IVT mRNA. are the most studied among delivery systems for IVT mRNA. FigureFigure 1. 1. IntracellularIntracellular barriers barriers for for inin vitro vitro transcribedtranscribed (IVT) (IVT) mRNA mRNA delivery: delivery: (1) (1) Interaction Interaction between between thethe delivery delivery system system and and cell cell membrane, membrane, (2) (2) endocyto endocytosis,sis, and and (3) (3) endosomal endosomal escape escape and and release release of of the the mRNAmRNA to to start start the the translation translation process. process.