TY - JOUR
AU - Avci-Adali, Meltem
AB - Abstract Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Reprogramming, Induced pluripotent stem cells, Gene expression, Gene delivery systems in vivo or in vitro, Direct cell conversion, Synthetic messenger RNA, Cell engineering Significance Statement The use of synthetic messenger RNA (mRNA) to produce desired proteins in cells and thereby reprogramming and transdifferentiation of cells is a very promising technology to enable the application of these engineered cells in clinic. Synthetic mRNA does not integrate into the genome and it is not necessary to enter the nucleus. The protein is directly produced in the cytosol. In this review, we summarize the progress of the synthetic mRNA-based cell engineering strategies and highlight the challenges, which have to be overcome to improve the application. Introduction The de novo synthesis of a missing or defective protein in targeted cells can be helpful for many applications in the field of regenerative medicine to repair, replace, and restore cells, tissues, or organs. Furthermore, the generation of patient-specific induced pluripotent stem cells (iPSCs) represents a promising source for cell-based therapies. In recent years, studies demonstrated the great potential of iPSCs in studying disease phenotypes and developing new drugs for therapy. Hereditary or acquired genetic disorders can lead to insufficient or dysfunctional biosynthesis of proteins and cause severe disease and even death. For example, cystic fibrosis and congenital surfactant protein B (SP-B) deficiency are rare genetic disorders, where the cystic fibrosis transmembrane conductance regulator (CFTR) or SP-B protein are defective or missing, which negatively affects the respiratory tract of the patient. Often, lung transplantation is the only way to save the patient's life. Other options are the development of gene therapies to produce the missing or defective proteins. Therefore, viral vector-based gene transfer strategies have been applied to correct the mutated CFTR [1]. In recent years, a promising alternative to viral vectors, for example, the exogenous delivery of in vitro transcribed (IVT) messenger RNA (mRNA) coding for desired proteins, has increased in importance [2, 3]. After exogenous delivery of IVT mRNA, cells start to produce functional proteins, which are naturally not synthetized or needed. Moreover, the transfer of therapeutic IVT mRNA into cells can also be used for vaccination to prevent tumor growth and infectious diseases. Thereby, the IVT mRNA-mediated synthesis of tumor cell or pathogen-specific antigens and the efficient activation of acquired immune system [4–6] can prevent tumor development and infectious diseases, or lead to improved eradication of tumor cells. Furthermore, stem cells and cells generated by directed differentiation can be engineered by IVT mRNA delivery to exhibit a new protein expression profile and to improve or alter cellular functions [7]. The induction of pluripotency in somatic cells to obtain iPSCs is a further important tool for the in vitro generation of autologous cells [8]. Using IVT mRNA containing 5-methylcytidine (5mC) and pseudouridine (ψ), Warren et al. [9] efficiently reprogrammed human fibroblasts into iPSCs by exogenous delivery of IVT mRNAs encoding transcription factors (Oct4, Sox2, Klf4, cMyc, and Lin28). Synthesis of Proteins in Cells Natural Pathway Since the genetic information of proteins cannot be decoded directly from DNA, the first step of natural protein synthesis is the transcription of the genetic information (DNA) into mRNA. Afterward, the mRNA is transported from the nucleus to sites of protein biosynthesis, the cytoplasmic ribosomes. Here, the mRNA is translated into polypeptide chains made of amino acids, which are then post-translationally modified and folded into their specific structure to form a functional protein. Synthetic Pathway Using commercially available kits, mRNA molecules can be easily synthesized from DNA templates containing the protein coding sequence by in vitro transcription. Simultaneously, the mRNA can be modified to increase stability and to reduce immunogenicity. Subsequently, the IVT mRNA is transfected into the cells to produce the desired protein. In the cytosol, the IVT mRNA is then directly translated into proteins by ribosomes. Advantages and Challenges of the Exogenous Delivery of IVT mRNA Many approaches aiming at the initiation of exogenous protein expression in cells use DNA- or RNA-based viral and nonviral vectors, such as retroviruses [8], adenoviruses [10], piggyBac transposons [11], Epstein-Barr virus-based episomal plasmids [12], Sendai viruses (SeV) [13], synthetic self-replicating RNA replicons [14], or IVT mRNAs [9] for gene transfer. However, due to the integration of transgenes into the host genome in case of retroviral vectors and the resulting high risk of permanent mutagenesis, there are safety concerns against the use of genome integrating vectors for human therapies [15–17]. Furthermore, the residual expression and reactivation of exogenously delivered transgenes [18], and immunogenicity [19] are additional problems related to integrating vectors [20], which limit their clinical application. To reduce or avoid integration of transgenes, several nonintegrative vectors (e.g., RNA viruses, circular plasmids, RNAs) and excision methods (Cre-Lox system [21], piggyBac transposons [11]) are used to produce the desired proteins in cells. Compared to genome integrating vectors, the main advantage of exogenously delivered IVT mRNAs is that the mRNA molecules do not insert into the host genome. Therefore, no oncogenic mutations are triggered. IVT mRNA is degraded after 2-3 days [9, 22, 23] in the cytoplasm and, due to its short half-life, no inactivation or excision of mRNA is necessary compared to protein expression strategies using genome integrating viral vectors. A further advantage of IVT mRNAs in comparison with other nonintegrative methods such as episomal DNA plasmids is their high reprogramming efficiency [24, 25]. Due to the large size of chemically complexed DNA plasmids, the transfection of cells is less efficient than with respectively packed IVT mRNA molecules [26]. It is also beneficial that mRNAs are transfected into the cytoplasm of cells, where they are directly translated into therapeutic target proteins without the need to enter the nucleus. DNA plasmids enter the nucleus preferably during the mitotic state, thus, the most effective protein expression occurs in dividing cells. In contrast, the mRNA-mediated protein expression takes place in dividing and nondividing cells, which is a further advantage of this method. The replication of SeV is also independent of nuclear factors and does not involve a DNA phase [27]. Hitherto, the reprogramming with SeV vector is still more efficient, easier, and cheaper than with IVT mRNAs and this method has been more successfully applied in independent laboratories [24, 28]. However, we believe that the improvements in IVT mRNA research and the optimization of the mRNA production and delivery, especially in the recent years, will further increase the efficiency of reprogramming and simplify the IVT mRNA method in the future, which has enormous potential for clinical applications. Theoretically, there is no limitation in size for the generation of the desired target mRNA sequence, and its production can be achieved at needed scales with commercially available materials. Furthermore, the use of mRNAs as therapeutic is beneficial, as they are of biological origin. Thus, naturally transient and cytosolic active IVT mRNA molecules are considered to be a safer and more potent alternative to most of the viral DNA/RNA vectors used for clinical applications. However, for some applications, such as cellular reprogramming experiments, transient protein expression may be a disadvantage of IVT mRNA. Thus, repeated daily transfections are required, which are time-consuming. Additionally, individual optimization of the transfection protocol for each cell type is needed for efficient protein synthesis. Another challenge is the production of IVT mRNA in large amounts with high reproducible quality under good manufacturing practice (GMP) conditions, which is required for therapeutic approaches like cell-based therapies. Therefore, the use of synthetic mRNA is still expensive. However, GMP production costs are approximately fivefold to tenfold lower for IVT mRNA than for recombinant proteins produced in eukaryotic cells [29]. Once GMP production is established, several grams of IVT mRNA can be manufactured per batch [29]. Furthermore, in the last few years, mRNA delivery methods, transfection reagents, and the mRNA molecule itself have been optimized to obtain more stability and translation efficiency and less immunogenicity [9, 30, 31], and now several IVT mRNAs are further on the march to clinical relevant applications. Delivery of IVT mRNA into Cells To produce appropriate amounts of the desired protein in cells, first, it is necessary to deliver the synthetic mRNA into the cytoplasm of the target cells. Because IVT mRNA molecules are negatively charged hydrophilic molecules, their uptake into cells is prevented. Thus, an efficient IVT mRNA delivery system is required to deliver synthetic mRNA into cells. For an effective transfection, the delivery system should fulfill several functions: (i) bind mRNA to form complexes, (ii) promote cellular uptake, (iii) protect mRNA from intracellular and extracellular nuclease degradation, and (iv) enable the release of mRNA into the cytoplasm [32]. Often, the transfection of cells is performed via the generation of complexes using cationic lipid-based transport vehicles (e.g., lipofectamine or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) [33] and the negatively charged mRNA molecules, called lipoplexes. Thereby, positively charged lipoplexes can be generated, which protect the IVT mRNA against extracellular degradation by RNases and associate with the negatively charged cell surface to promote cellular entry via natural endocytosis. Studies have demonstrated the internalization of synthetic mRNA by clathrin- and caveolin-mediated endocytosis and the involvement of scavenger receptors [34, 35]. The vesicles containing the mRNA transfection complexes are then directed to late endosomes. Before their fusion with lysosomal vesicles, transfection complexes escape from the late endosomes into the cytosol (Fig. 1). Furthermore, other endocytosis-mediating cationic carriers, such as polyethylenimine (PEI), poly(l-lysine), or dendrimers, can also be used for the generation of polyplexes to deliver IVT mRNA into cells. Another common method to deliver mRNA into cells is electroporation [36]. Here, the cell membrane lipid double layer is temporarily destabilized and the mRNAs enter the cells across the spontaneously formed pores after applying a high electric field pulse to the cells. A major drawback of this method is that numerous cells die during this procedure. However, electroporation seems to work better than lipid-based transfection for some cell types (e.g., blood cells). Figure 1 Open in new tabDownload slide Schematic overview of (I) synthesis of IVT mRNA and (II) delivery of IVT mRNA into cells. In the PCR, the DNA template containing the coding DNA sequence of the desired protein is generated with a 5′-T100-250 overhang from the corresponding DNA plasmid. To produce the mRNA, an in vitro transcription reaction is performed. During in vitro transcription, a 3′-poly(A) tail is generated to prevent the mRNA from nuclease degradation. Additionally, mRNA can be generated using modified nucleosides (e.g., 5-methylcytidine and pseudouridine) and a 5′-cap structure (e.g., ARCA) to improve translation and mRNA stability. The presence of RNAse inhibitor during the in vitro transcription protects the mRNA from nuclease attack. To transfect cells, mRNA molecules are complexed with a cationic lipid-based transport vehicle. The complexes are taken up via endocytosis. After endosomal escape of the IVT mRNA into the cytoplasm, the mRNAs are translated by ribosomes into the desired protein(s). Abbreviations: ARCA, anti-reverse cap analog; IVT, in vitro transcribed; mRNA, messenger RNA; PCR, polymerase chain reaction. Figure 1 Open in new tabDownload slide Schematic overview of (I) synthesis of IVT mRNA and (II) delivery of IVT mRNA into cells. In the PCR, the DNA template containing the coding DNA sequence of the desired protein is generated with a 5′-T100-250 overhang from the corresponding DNA plasmid. To produce the mRNA, an in vitro transcription reaction is performed. During in vitro transcription, a 3′-poly(A) tail is generated to prevent the mRNA from nuclease degradation. Additionally, mRNA can be generated using modified nucleosides (e.g., 5-methylcytidine and pseudouridine) and a 5′-cap structure (e.g., ARCA) to improve translation and mRNA stability. The presence of RNAse inhibitor during the in vitro transcription protects the mRNA from nuclease attack. To transfect cells, mRNA molecules are complexed with a cationic lipid-based transport vehicle. The complexes are taken up via endocytosis. After endosomal escape of the IVT mRNA into the cytoplasm, the mRNAs are translated by ribosomes into the desired protein(s). Abbreviations: ARCA, anti-reverse cap analog; IVT, in vitro transcribed; mRNA, messenger RNA; PCR, polymerase chain reaction. Immune Responses to IVT mRNA In eukaryotic cells, pattern recognition receptors (PRRs) of the innate immune system including Toll-like receptors (TLR) 3 [37, 38], 7 [39–42], and 8 [41], as well as RIG-(retinoic acid-inducible gene)-I like receptors (RLRs) recognize foreign single stranded (ss) and double stranded (ds) RNA molecules (Table 1) and transduce signals to induce a nuclear factor-κB (NF-κB) and type I interferon (IFN) mediated viral immune response. The signaling through cytoplasmic RLRs and the endosomal TLR3 and TLR7/8 receptors activate the downstream transcription factors interferon-regulatory factor (IRF)3, IRF7, and NF-κB [53]. Thereby, the expression of pro-inflammatory cytokines and type I IFNs (IFN-α and IFN-β) is induced. Table 1 List of pattern recognition receptors (PRRs) detecting foreign RNA molecules PRR . Ligand . References . TLR3 dsRNA [37, 38] TLR7 ssRNA [39–42] TLR8 ssRNA [41] RIG-I 5′-Triphosphate (5′ppp) and 5′-diphosphate (5′pp) bearing dsRNA, dsRNA with cap0 5′-end (7-methylguanosine cap linked to the mRNA through a 5′-5′- triphosphate bridge without 2′O-methylation at the N1 position: m7G5′ppp5′N1) [43–47] MDA5 Long dsRNA [48–50] NLRP3 dsRNA [51] NOD2 ssRNA [52] PRR . Ligand . References . TLR3 dsRNA [37, 38] TLR7 ssRNA [39–42] TLR8 ssRNA [41] RIG-I 5′-Triphosphate (5′ppp) and 5′-diphosphate (5′pp) bearing dsRNA, dsRNA with cap0 5′-end (7-methylguanosine cap linked to the mRNA through a 5′-5′- triphosphate bridge without 2′O-methylation at the N1 position: m7G5′ppp5′N1) [43–47] MDA5 Long dsRNA [48–50] NLRP3 dsRNA [51] NOD2 ssRNA [52] Abbreviations: MDA5, melanoma differentiation-associated protein 5; mRNA, messenger RNA; N1, nucleotide 1; NLRP3, NLR family, pyrin domain containing 3; NOD2, nucleotide-binding oligomerization domain-containing protein 2; RIG-I, retinoic acid-inducible gene-1; TLR, toll-like receptor. Open in new tab Table 1 List of pattern recognition receptors (PRRs) detecting foreign RNA molecules PRR . Ligand . References . TLR3 dsRNA [37, 38] TLR7 ssRNA [39–42] TLR8 ssRNA [41] RIG-I 5′-Triphosphate (5′ppp) and 5′-diphosphate (5′pp) bearing dsRNA, dsRNA with cap0 5′-end (7-methylguanosine cap linked to the mRNA through a 5′-5′- triphosphate bridge without 2′O-methylation at the N1 position: m7G5′ppp5′N1) [43–47] MDA5 Long dsRNA [48–50] NLRP3 dsRNA [51] NOD2 ssRNA [52] PRR . Ligand . References . TLR3 dsRNA [37, 38] TLR7 ssRNA [39–42] TLR8 ssRNA [41] RIG-I 5′-Triphosphate (5′ppp) and 5′-diphosphate (5′pp) bearing dsRNA, dsRNA with cap0 5′-end (7-methylguanosine cap linked to the mRNA through a 5′-5′- triphosphate bridge without 2′O-methylation at the N1 position: m7G5′ppp5′N1) [43–47] MDA5 Long dsRNA [48–50] NLRP3 dsRNA [51] NOD2 ssRNA [52] Abbreviations: MDA5, melanoma differentiation-associated protein 5; mRNA, messenger RNA; N1, nucleotide 1; NLRP3, NLR family, pyrin domain containing 3; NOD2, nucleotide-binding oligomerization domain-containing protein 2; RIG-I, retinoic acid-inducible gene-1; TLR, toll-like receptor. Open in new tab RLRs, consisting of RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), can detect foreign RNA in the cytoplasm of most cell types. After RNA sensing, RIG-I and MDA5 initiate downstream signaling, which leads to type I IFN gene expression and pro-inflammatory cytokines. LGP2 appears to have a regulatory role in RLR signaling; however, its precise action is still to be determined [54]. Andries et al. [55] demonstrated in their studies that, in addition to TLR3-mediated recognition of IVT mRNA, nucleotide-binding domain (NOD)-like receptors (NLRs) are also involved in synthetic mRNA detection. NLRs regulate caspase-1 production, which plays essential roles in apoptosis (programmed cell death). Thus, the overexpression of caspase-1 after IVT mRNA delivery resulted in cell death and inflammation. Especially NLRP3 [51] and NOD2 [52], which are members of the NLRs, have been found to sense RNA. The type I IFNs bind to IFN receptors of activated cells and neighboring cells and lead to the generation of IFN-stimulated gene factor 3 (ISGF-3) that initiates the transcription of more than 300 IFN-stimulated genes (ISGs) [53], including dsRNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetases (OASs), and RNA-specific adenosine deaminase (ADAR), which are produced to intensify antiviral activity. The activation of PKR by dsRNA leads to the inhibition of mRNA translation and thereby to the reduction of protein synthesis [56], which is naturally required to suppress viral replication. Furthermore, the transcription factor NF-κB can be activated and cellular apoptosis can be induced. The activation of OASs by dsRNA leads to the cleavage of ssRNA. The activation of ADARs, also by dsRNA, results in RNA editing by conversion of adenosine (A) to inosine (I). Consequently, the formation of weak I:U mismatches can destabilize RNA and reduce the coding capacity of mRNA. In cellular reprogramming and lineage-conversion experiments, specific reprogramming factors have to be expressed over a period of several days. Since mRNA is degraded in the cytoplasm relatively quickly, synthetic mRNA-mediated protein expression lasts only a few hours to 1-3 days [9, 23, 57]. Therefore, the delivery of IVT mRNA molecules is required every day to maintain a continuous protein level, which is crucial for the success of the experiment. However, cells are more stressed than after a single mRNA transfection due to the repeated activation of the innate immune response against synthetic mRNA molecules and the transfection reagent, which results in excessive cell death. Thus, only the suppression of this innate immune response, for example, by adding immunosuppressive molecules such as B18R protein, allows the repeated transfection of cells with IVT mRNA during iPSC generation [9]. Angel and Yanik [57] also showed that combined siRNA-induced knockdown of innate immune response mediators (Ifnb1, Eif2ak2, and Stat2) blocked the immune reaction and enhanced cell survival after repeated treatment with mRNAs for reprogramming. Thereby, the fibroblasts, which were repeatedly cotransfected with siRNA and mRNA (five times at 48 hours intervals) were prevented from extensive cell death and the expression of reprogramming factors could be maintained over several days (up to 5 days) through multiple rounds of cell division. However, for other applications, like vaccination with synthetic mRNA, the induction of an immune response could be beneficial, as mRNA can additionally work as an adjuvant by enhancing immunological response and antigen presentation [58]. Modifications of Synthetic mRNAs The use of mRNA for protein replacement is not a new concept, but it was also not very popular for many years, because of the molecule's instability and immunogenicity. About 10 years ago, Kariko's group provided a breakthrough in mRNA therapy by incorporation of (naturally occurring) modified nucleosides into the mRNA [59]. During the last few years, modifications of synthetic mRNA (Fig. 2) and the optimization of delivery methods have been shown to facilitate the use of mRNA for therapeutic applications. Figure 2 Open in new tabDownload slide Schematic structure of in vitro transcribed messenger RNA (mRNA). Modifications of mRNA, such as adding a synthetic cap analog at the 5′-end and a poly(A) tail at the 3′-end, as well as incorporation of modified nucleosides (e.g., 5-methylcytidine or pseudouridine), improve the stability of synthetic mRNAs, increase their translational activity in cells, and reduce their immunogenicity. Figure 2 Open in new tabDownload slide Schematic structure of in vitro transcribed messenger RNA (mRNA). Modifications of mRNA, such as adding a synthetic cap analog at the 5′-end and a poly(A) tail at the 3′-end, as well as incorporation of modified nucleosides (e.g., 5-methylcytidine or pseudouridine), improve the stability of synthetic mRNAs, increase their translational activity in cells, and reduce their immunogenicity. In eukaryotic cells, mRNA molecules naturally bear a cap structure at the 5′ end and a long sequence of polyadenylate residue (poly(A) tail) at the 3′ end that are added after transcription of the DNA. In contrast, the standard IVT mRNAs without modifications contain a 5′-triphosphate structure at the 5′ end and no poly(A) tail. As the cap structure and the poly(A) tail of the mRNA greatly enhance the stability of natural mRNA, synthetically produced mRNA can also be modified to contain these structures. Using these modifications, the stability of synthetic mRNAs can be improved, immunogenicity can be reduced, and the translation efficiency can be increased. The incorporation of modified nucleosides, for example, 5mC and Ψ instead of cytidine (C) and uridine (U), resulted in reduced immunogenicity and promoted the mRNA stability [31, 56, 59–62]. Using this method, cell death and toxicity caused by synthetic mRNA are reduced and ectopic protein expression is increased. In Table 2, diverse IVT mRNA molecules generated using modified nucleosides are listed to produce respective proteins. Warren and colleagues demonstrated the successful applicability of IVT mRNAs with modified nucleosides for the generation of iPSCs [9]. Here, cytidine and uridine were completely (100%) replaced by 5mC and Ψ. Further studies showed that a reduced amount of both modified nucleosides (25% of 5mC and Ψ [14] or 25% of 5mC and 2-thiouridine (2sU) [3, 63]) or the incorporation of one type of modified nucleoside (100% Ψ [7, 67] or 100% N-1-methylpseudouridine (m1Ψ) [68, 69]) into mRNA is also beneficial for an efficient protein synthesis compared to IVT mRNA with unmodified nucleosides. Recently, Andries et al. [68] showed that the incorporation of m1Ψ in combination with 5mC further improved cellular viability and protein expression, and reduced immunogenicity of luciferase mRNA in various mammalian cell lines, A549 cells (human lung epithelial cells), C2C12 cells (murine myoblasts), HeLa cells (human cervix epithelial cells), BJ fibroblasts (from newborn human foreskin), and human primary keratinocytes (from neonatal foreskin) compared to 5mC and Ψ modified mRNA. Furthermore, templates for in vitro transcription can be optimized by the incorporation of 5'- and 3'- terminal untranslated regions (UTRs), such as alpha- and beta-globin UTRs [71], to enhance RNA stability and translational efficiency. Additionally, the presence of a strong Kozak translation initiation sequence in the 5'-UTR [72] of mRNA increases the translational capacity. Moreover, a 5'-cap-analog, for example, the anti-reverse cap analog (ARCA, 3´-O-Me-m7G(5´ )ppp(5´ )G), can be added to mRNAs to prolong their half-life in the cytoplasm and to promote the binding of synthetic mRNA to the small ribosomal subunit, which in turn improves the translation of the mRNA into the desired protein [73–76]. However, after IVT, the produced mRNA still contains significant amounts of uncapped ssRNA and short dsRNA fragments, bearing a triphosphate at the 5´-end (5´ppp) that can lead to RIG-I activation. Although RIG-I was initially identified as an ssRNA sensor [77, 78], subsequent studies demonstrated that the activation of RIG-I requires dsRNA [43, 44]. Further studies revealed that dsRNA with 5'-diphosphates [45] as well as cap0 (m7G5'ppp5'N1) [46, 47] are also able to bind and activate RIG-I. Additional to the 5´-end modification of the IVT mRNA, a poly(A) tail (100-250 adenylates) as in the case of natural mRNA can be added at the 3‘-UTR of mRNAs to protect them from nuclease degradation [73, 79]. However, despite all these modifications, IVT mRNA can still induce a type-I interferon-dependent immune response via PRR recognition [57, 80, 81]. Table 2 Insertion of different amounts of modified nucleosides into IVT mRNA molecules, which have been shown to be beneficial for the expression of different proteins Modification . Expressed proteins . References . 100% of 5mC and Ψ Reprogramming factors, VEGF-A, PSGL-1/SLeX/IL-10, MyoD1, HSV1-tk, nuclease, luciferase [7, 9, 25, 63–66] 100% of Ψ Erythropoietin, PSGL-1/SLeX/IL-10, luciferase [7, 67] 100% of 5mC and m1Ψ or m1Ψ alone Firefly luciferase [68, 69] 25% of 5mC and 2sU Erythropoietin, RFP, nuclease [3, 63] 25% of 5mC and Ψ B18R interferon inhibitor [14] 10% of 5mC and 2sU Foxp3 [70] Modification . Expressed proteins . References . 100% of 5mC and Ψ Reprogramming factors, VEGF-A, PSGL-1/SLeX/IL-10, MyoD1, HSV1-tk, nuclease, luciferase [7, 9, 25, 63–66] 100% of Ψ Erythropoietin, PSGL-1/SLeX/IL-10, luciferase [7, 67] 100% of 5mC and m1Ψ or m1Ψ alone Firefly luciferase [68, 69] 25% of 5mC and 2sU Erythropoietin, RFP, nuclease [3, 63] 25% of 5mC and Ψ B18R interferon inhibitor [14] 10% of 5mC and 2sU Foxp3 [70] Abbreviations: Ψ, pseudouridine; 2sU, 2-thiouridine; 5mC, 5-methylcytidine; Foxp3, forkhead box P3; HSV1-tk, herpes simplex virus 1-thymidine kinase; IL-10, interleukin 10; IVT, in vitro transcribed; m1Ψ, N-1-methyl-pseudouridine; mRNA, messenger RNA; MyoD1, myogenic differentiation factor 1; PSLG-1, P-selectin glycoprotein ligand-1; RFP, red fluorescent protein; SLeX, Sialyl Lewisx; VEGF-A, vascular endothelial growth factor-A. Open in new tab Table 2 Insertion of different amounts of modified nucleosides into IVT mRNA molecules, which have been shown to be beneficial for the expression of different proteins Modification . Expressed proteins . References . 100% of 5mC and Ψ Reprogramming factors, VEGF-A, PSGL-1/SLeX/IL-10, MyoD1, HSV1-tk, nuclease, luciferase [7, 9, 25, 63–66] 100% of Ψ Erythropoietin, PSGL-1/SLeX/IL-10, luciferase [7, 67] 100% of 5mC and m1Ψ or m1Ψ alone Firefly luciferase [68, 69] 25% of 5mC and 2sU Erythropoietin, RFP, nuclease [3, 63] 25% of 5mC and Ψ B18R interferon inhibitor [14] 10% of 5mC and 2sU Foxp3 [70] Modification . Expressed proteins . References . 100% of 5mC and Ψ Reprogramming factors, VEGF-A, PSGL-1/SLeX/IL-10, MyoD1, HSV1-tk, nuclease, luciferase [7, 9, 25, 63–66] 100% of Ψ Erythropoietin, PSGL-1/SLeX/IL-10, luciferase [7, 67] 100% of 5mC and m1Ψ or m1Ψ alone Firefly luciferase [68, 69] 25% of 5mC and 2sU Erythropoietin, RFP, nuclease [3, 63] 25% of 5mC and Ψ B18R interferon inhibitor [14] 10% of 5mC and 2sU Foxp3 [70] Abbreviations: Ψ, pseudouridine; 2sU, 2-thiouridine; 5mC, 5-methylcytidine; Foxp3, forkhead box P3; HSV1-tk, herpes simplex virus 1-thymidine kinase; IL-10, interleukin 10; IVT, in vitro transcribed; m1Ψ, N-1-methyl-pseudouridine; mRNA, messenger RNA; MyoD1, myogenic differentiation factor 1; PSLG-1, P-selectin glycoprotein ligand-1; RFP, red fluorescent protein; SLeX, Sialyl Lewisx; VEGF-A, vascular endothelial growth factor-A. Open in new tab Since the protein expression by IVT mRNA is transient, one clear disadvantage of mRNA-based reprogramming is that repeated daily mRNA transfections are required over several days for continuous expression of reprogramming factors. Thus, the repression of immunogenicity against exogenously delivered mRNA and the increased stability of mRNA molecules are especially important for efficient reprogramming of cells. Warren et al. [9] synthesized IVT mRNAs and treated fibroblasts daily with transfection complexes and additionally with an interferon inhibitor (B18R) to reduce the immune response during IVT mRNA-based reprogramming of cells. Thereby, cell viability was increased and iPSCs could be very efficiently generated using IVT mRNAs encoding the transcription factors Oct4, Sox2, Klf4, cMyc, and Lin28. Afterwards, these cells were directly differentiated into myogenic cells using MyoD mRNA, which contained 5mC and Ψ nucleosides to increase stability and translation of IVT mRNA. Another research group generated self-replicating RNA constructs for successful reprogramming of fibroblasts [14]. These very long RNA constructs (about 15,000 nucleotides long) are based on a single noninfectious (nonpackaging) and self-replicating Venezuelan Equine Encephalitis virus RNA replicon containing four open reading frames (Oct4, Sox2, Klf4 with either cMyc or Glis-1) for the induction of pluripotency [14]. Thus, each construct is able to encode for more than one protein. The self-replication of RNA constructs in cells makes the daily transfection with mRNA unnecessary, but it requires the addition of conditioned medium containing B18R. Recently, Poleganov et al. generated reprogramming mRNAs (Oct4, Sox2, Klf4, Lin28, cMyc, and Nanog) using nonmodified nucleosides and combined them with immune reaction circumventing mRNAs coding for E3, K3, and B18R from vaccinia virus and the enhancer microRNA miR302/367 to overcome RNA-related cytotoxicity during the reprogramming process [64]. Using this approach, nonmodified mRNAs, which are known to induce cellular defense mechanisms, were effectively able to convert fibroblasts into iPSCs with four repeated transfections within 11 days and blood-outgrowth endothelial progenitor cells were reprogrammed within 10 days with only eight daily transfections. After in vitro transcription, the product may contain undesired RNA molecules in addition to IVT mRNA, such as truncated RNAs due to incomplete synthesis, abortive transcription, uncapped mRNAs, and dsRNA. Thus, an additional method that could help to reduce the immunogenicity of synthetic mRNA is high-performance liquid chromatography purification. Thereby, short dsRNA fragments and other contaminants that cause immune reactions can be eliminated after in vitro transcription reaction [30]. Moreover, for efficient transfection and continuous protein biosynthesis, the cell quality and density at the beginning of mRNA treatments are as important as the administered IVT mRNA dose per cell. Differences in mRNA transfection efficiency are expected for cells of different origins, due to their different transfectability. Thus, the choice of transfection reagent is also important and should be tested to achieve the best mRNA transfection efficiency for each cell type and application [82]. Applications of IVT mRNA In the last few years, mRNA engineering has become more and more popular. There are promising applications not only in the field of regenerative medicine. Besides reprogramming, (trans)differentiation and stem cell homing, tumor treatment, vaccination, and the production of growth factors are also auspicious mRNA-based approaches (Fig. 3). Figure 3 Open in new tabDownload slide Perspectives of in vitro transcribed (IVT) mRNA engineered cells. Exogenous delivery of IVT mRNA molecules for reprogramming somatic cells into induced pluripotent stem cells, directed (trans)differentiation of cells in desired cell types (e.g., myocytes), production of secretable proteins in cells, overexpression of receptors on mesenchymal stem cells to improve homing behavior, vaccination against cancer cells, or repair of gene defects in diseased cells. Abbreviation: mRNA, messenger RNA. Figure 3 Open in new tabDownload slide Perspectives of in vitro transcribed (IVT) mRNA engineered cells. Exogenous delivery of IVT mRNA molecules for reprogramming somatic cells into induced pluripotent stem cells, directed (trans)differentiation of cells in desired cell types (e.g., myocytes), production of secretable proteins in cells, overexpression of receptors on mesenchymal stem cells to improve homing behavior, vaccination against cancer cells, or repair of gene defects in diseased cells. Abbreviation: mRNA, messenger RNA. Reprogramming and (Trans)Differentiation of Cells Using IVT mRNA In 2006, Yamanaka and Takahashi [8] accomplished a groundbreaking discovery in stem cell research by reprogramming of adult somatic cells in iPSCs using a specific set of transcription factors, Oct4, Sox2, Klf4 and cMyc (OSKM), also called the Yamanaka factors. Until then, embryonic stem cells (ESCs) were the only pluripotent stem cell source. Thus, these iPSCs are a promising alternative to ESCs, as they are generated without the destruction of an embryo and the donor-specific isolation of adult somatic cells is classified as ethically safe. A preliminary report of the first human trial where ESC-derived retinal pigment epithelium (RPE) cells were transplanted into patients with Stargardt's Macular Dystrophy and dry age-related macular degeneration (AMD) showed beneficial effects [83]. There were no signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection during the first 4 months under immunosuppressive therapy, which started one week before surgery and continued for 12 weeks after transplantation. This cell-based therapy also seemed to improve the vision of treated patients. However, several systemic adverse effects were observed in patients, which seemed to be related to the immunosuppressive therapy [84]. Thus, the transplantation of RPE cells derived from autologous iPSCs could circumvent the need for immunosuppressive drugs due to their donor-specific origin. In 2014, Japan started the first clinical trial using RPE cells derived from iPSCs for treatment of AMD under the supervision of Masayo Takahashi [85]. Fibroblasts from a 70-year-old woman were reprogrammed and subsequently differentiated into RPE cells to form an autologous monolayer, which was then transplanted into the subretinal space, between the degenerating photoreceptors and the RPE, of the AMD patient's eye. Prior to this clinical trial, the group examined the genetic stability of the generated cells regarding mutations that can occur in the reprogramming process and found that there was no tumor growth after implantation in immunodeficient NOG mice [86]. Due to the patient's advanced disease state, it is expected that the patient's vision would not be completely restored [87]. However, the results will hopefully show whether potential side effects, such as immune rejection or cancerous growth, can be avoided in comparison to ESC-derived cell transplants, as shown in earlier trials [83]. Thus, this initial human study provides new hope for the application of iPSC-derived cells in regenerative medicine. Often, human iPSCs contain genomic integrations or viral vector contamination due to the applied reprogramming method. Thus, the tumorigenic potential of such pluripotent cells represents a considerable risk for clinical applications [88]. To avoid these unwanted alterations of cells and thereby mutations, nonintegrative and nonviral methods, such as exogenous delivery of IVT mRNA [24], are highly recommended to obtain clinically applicable iPSCs and desired cells from them. It was demonstrated that mRNA-derived iPSCs more faithfully recapitulated the global transcriptional signature of human ESCs than retrovirus-derived iPSCs [9]. Furthermore, Steichen et al. [16] compared the genomic integrity of retrovirus-derived iPSCs with that of mRNA-derived iPSCs. Single nucleotide polymorphism analysis of mRNA-derived iPSCs did not differ from the original source (parental fibroblast), whereas retrovirus-derived iPSCs did. The occurrence of copy number variations seemed to be clone-dependent and independent of the reprogramming method. This shows that mRNA-based technology appears to be safer for clinical applications than retrovirus-derived iPSCs. However, iPSC lines generated by IVT mRNAs should also be carefully analyzed regarding genome integrity before using them for clinical applications. Huangfu et al. [89] were also able to reprogram human fibroblasts by the overexpression of only two factors, Oct4 and Sox2, by simultaneous addition of valproic acid, a histone deacetylase inhibitor. This shows that not only the gene expression, but also epigenetic processes, such as chromatin modifications, plays an important role in cellular reprogramming and that efficiency and kinetics can be improved by adding epigenetic modulators. Another molecule that has been shown to accelerate nuclear reprogramming processes by modification of the chromatin structure is the myogenic differentiation protein MyoD [90]. Different studies have shown that the fusion of a strong transcriptional active fragment of MyoD to the Oct4 transcription factor leads to improved chromatin accessibility and enhanced recruitment of chromatin remodeling proteins and transcription factors to pluripotency genes, which results in radical acceleration of reprogramming [90, 91]. Furthermore, Liu et al. [92] demonstrated that the sequential introduction of reprogramming factors (Oct4 and Klf4 first, then cMyc, and finally Sox2) using a viral vector outperforms the classical reprogramming protocol in which the transcription factors are delivered simultaneously. Thereby, a time-sensitive requirement of individual factors for optimal reprogramming was demonstrated, which could be also applied for mRNA-based reprogramming. The delivery of IVT mRNA in cells could also be auspicious for the direct differentiation of somatic cells (fibroblasts) into a desired cell type, a process called lineage conversion or transdifferentiation [28, 93]. For example, mesodermal cells such as myocytes [94, 95] (smooth muscle cells, cardiomyocytes) and blood cells [96], endodermal cells (pancreatic cells, hepatocytes) [97–99] and ectodermal cells (neurons) [100] are interesting cell types for cell-based therapies in regenerative medicine. Using IVT mRNAs encoding HNF1A plus any two of the factors FOXA1, FOXA3, or HNF4A, human fibroblasts were directly converted into hepatocyte-like cells [99]. Recently, Preskey et al. [65] showed the successful transdifferentiation of HFFs into myoblast-like cells in only 7 days and with only four daily transfections with IVT mRNA encoding the myogenic differentiation factor MyoD1, without leaving a genomic footprint. This direct reprogramming method seems to be a gentler and faster approach then the initial generation of iPSCs and subsequent differentiation into the desired cell type. Furthermore, it could avoid increased accumulation of genetic and epigenetic aberrations. Therefore, the mRNA-mediated (trans)differentiation technology to create integration-free and virus-free myocytes could be beneficial to cell-based clinical applications. During the reprogramming process with IVT mRNA, the acceptance of daily transfection protocols is poor. Thus, researchers aim to prolong mRNA-mediated protein expression, for instance with synthetic self-replicating RNAs [14] or self-assembled mRNA nanoparticles (mRNA-NPs) [101]. The optimization of transfection protocols for mRNA-based reprogramming and transdifferentiation is an ongoing process and holds significant potential for biomedical research and regenerative medicine, which will certainly be better accessible to more research labs in the future. Tumor Treatment For adoptive immunotherapy of hematologic and solid malignancies, cytotoxic T lymphocytes can be modified with chimeric antigen receptors (CARs) that lead to both antigen binding and T cell activation to specifically target tumor antigens on the cell surface. To further enhance T cell potency and specificity, CARs can be combined with costimulatory ligands (e.g., CD80, CD86), chimeric costimulatory receptors (e.g., CD3, CD28), and cytokines (IL-15, IL-12) [102]. CARs have been shown to be effective in preclinical models and are being tested in several clinical trials [103]. Different research groups have generated target-specific CAR mRNAs to attack tumor cells. For example, T cells with an exogenously introduced anti-CD19 CAR mRNA were potent and specific killers of CD19 positive cells (human erythromyeloblastoid leukemia cells expressing CD19). Interestingly, significant prolongation of survival was shown after a single injection of the T cells expressing anti-CD19 CAR in a leukemia xenograft mouse model [104]. In another study, mesothelin-specific CAR mRNA transfected T cells (CARTmeso) persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites [105]. The antitumor immune response was demonstrated in both CARTmeso cells and in the two first-in-human case reports of patients with mesothelin expressing solid malignancies. Recently, IVT mRNA was also used to generate T cells transiently expressing CD33-specific CAR (CART33), which showed potent preclinical activity against human acute myeloid leukemia [106]. One further mRNA-based approach of targeted cancer therapy is the systemic delivery of IVT mRNA coding for the suicide gene herpes simplex virus 1-thymidine kinase (HSV1-tk) [66]. In this study, Wang and colleagues showed that HSV1-tk mRNA formulated into lipid-protamine-RNA nanoparticles (LPR-NPs) was taken up by human lung carcinoma cells (NCI-H460) and was subsequently translated in vitro. After the intravenous administration of HSV1-tk mRNA containing LPR-NPs in an H460 xenograft-bearing nude mouse model, an anticancer effect was detected in vivo by the induction of apoptosis and the inhibition of tumor growth. In this study, the LPR-NPs were used to improve the efficiency of IVT mRNA delivery for gene therapy in vivo. The NPs containing mRNA also revealed to be significantly more effective in suppressing tumor growth compared to NPs formulated with plasmid DNA [66]. Treatment of Genetic Defects A novel alternative for the treatment of the genetic disorder cystic fibrosis is the mRNA-mediated gene replacement therapy. Bangel-Ruland et al. showed in their study that CFTR mRNA delivery in a human bronchial cystic fibrosis cell line and in primary human nasal epithelial cells resulted in CFTR protein synthesis and the proper integration of CTFR into the cell membrane [2]. Thereby, impaired CTFR function could be restored in transfected cells. Kormann et al. also successfully applied IVT mRNA as a tool to repair gene defects in a rare genetic disease [3]. SP-B, which is deficient in lethal congenital lung disease cells, was produced after the local application of an aerosol of SP-B mRNA to the lungs of mice with lethal congenital lung disease with life-saving therapeutic efficacy. Even the systemic expression of erythropoietin (Epo) could be achieved by intraperitoneal or intramuscular injection of a synthetic Epo mRNA [3, 67]. In recent studies, Kormann's group assessed the time- and site-specific expression of mRNA encoding the regulatory T cell (Treg) transcription factor Foxp3, in order to treat or prevent diseases caused by an imbalance in helper T cell responses [70]. Foxp3 is the transcription factor that characterizes Tregs, which are capable of suppressing an overactive Th2 response. In allergic diseases like asthma, this regulatory mechanism is defective. The administration of Foxp3 mRNA via intratracheal spray rebalanced pulmonary T helper cell responses and was protective against asthma symptoms in a mouse model. This protection was conferred following delivery of Foxp3 mRNA either before or after the onset of allergen challenge, demonstrating its potential for both as a preventive and as a therapeutic agent [70]. Recently, Michel et al. demonstrated that the exogenous delivery of IVT mRNA can be also used to produce proteins for the treatment of patients with single-gene mutations that result in a missing or defective protein, like in alpha-1-antitrypsin (AAT) deficiency disorder [107]. AAT, which is normally produced in the liver and released by activated neutrophil granulocytes and macrophages, plays an important role in the prevention of proteolytic damage in host tissue during inflammatory processes. They showed that the delivered AAT mRNA was functionally expressed in the transfected A549 lung carcinoma cells and HEK293 (human embryonic kidney) cells. The functionality of the produced AAT protein was demonstrated by its inhibitory effect on neutrophil elastase. The inhibition of elastase activity revealed that the produced AAT in cells was functional and, therefore, this approach is encouraging as a new treatment strategy. Vaccination One additional pioneering approach is the vaccination against viral infections and cancers using IVT mRNAs. Most of these investigations have focused on autologous dendritic cells (DCs) that are transfected with IVT mRNA either in vitro for adoptive transfer into the patient or in vivo by direct administration, in order to express specific anti-tumor or anti-viral antigens. Thereby, major histocompatibility complex class I and class II on DCs present the antigen in order to induce an immune response against the tumor or the pathogens. Lorenzi et al. [42] showed that the intranasal application of naked IVT mRNA coding for the 65 kDa heat shock protein (Hsp65), which is one of the major immune reactive proteins during Mycobacterium tuberculosis infection, led to the protection against tuberculosis (TB) in an experimental mouse model in vivo. Hsp65 mRNA was able to reach lung antigen presenting cells (APCs; mainly DCs) after intranasal vaccination and to induce the Th1 cytokines IFN-γ and TNF-α, which are essential for the development of protective immunity against TB. Only the immunized mice showed a significant decrease in bacterial counts after infection. Besides this intranasal delivery strategy, patients can also be treated with antigen-encoding mRNA that is directly injected into the patient's body (local or intravenous), and then taken up by APCs to induce a humoral and cellular immune response (Th1 and Th2) against the disease [108]. So far, the results have shown that mRNA-based vaccines induce humoral and cellular immune responses and result in an increased survival rate. To date, there are two interesting clinical trials in progress, one for castration-resistant prostate cancer [109] and another for nonsmall cell lung cancer [110]. Both studies showed that the application of RNActive® vaccines (from the company CureVac) to humans was safe [60]. Further promising mRNA-based vaccination projects are focusing on the development of vaccines against infectious diseases, like influenza A virus [5] and HIV-1 [4]. Petsch et al. [5] also demonstrated a further advantage of mRNA-based vaccines by showing that mRNA vaccines can be lyophilized and that they retain their full biological activity. This solves the problems regarding the thermal stability of the conventional vaccines, particularly in countries where the infrastructure makes it difficult to maintain the cold chain. Lyophilized mRNA-based vaccines were stable when exposed to thermal stress at temperatures of 25°C and 40°C for periods of several years or months, respectively. Stability could be shown even under extreme stress conditions at a temperature of 60°C for several months [60]. In Vivo Production of Secretable Proteins In the field of regenerative medicine, the approach of IVT mRNA-based expression of paracrine factors from mRNA transfected cells after in vivo transplantation or the direct administration of synthetic mRNA is another promising method for cellular reprogramming and the control of progenitor cell fate and homing. Zangi et al. [25] created an IVT mRNA encoding for the human vascular endothelial growth factor-A (VEGF-A) and injected the mRNA intramyocardially in a mouse myocardial infarction model. They demonstrated that the applied VEGF-A mRNA markedly improved heart function and enhanced the long-term survival of treated mice as a result of the IVT mRNA-mediated direct differentiation of heart progenitor cells toward cardiovascular cell types in vivo. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases. For example, patients suffering from inflammatory diseases, such as multiple sclerosis, could be treated with mRNA engineered MSCs to reduce local inflammation. Levy et al. [7] transfected MSCs with IVT mRNA to simultaneously express functional proteins (P-selectin glycoprotein ligand-1 [7] and sialyl Lewisx [SLeX]) and to induce the rapid homing of cells to inflamed tissues. After homing, the additional expression of the potent immunosuppressive cytokine interleukin-10 (IL-10) in MSCs transiently increased the levels of IL-10 in the inflamed region and showed an anti-inflammatory effect in vivo, which significantly reduced local inflammation. This shows that mRNA engineered stem cells can also be considered as vehicles for the simultaneous delivery of potent immunomodulatory factors to distant inflammation sites. Conclusion The application of IVT mRNA is a versatile and promising tool for the delivery of transgenes into desired cells. Due to its safety and high efficiency, the use of IVT mRNA is especially attractive for applications in regenerative medicine, vaccination, the treatment of hereditary diseases and tumors, and cell engineering. IVT mRNA can be produced in large-scale amounts in GMP quality, allowing the clinical application of mRNA-based therapies. Furthermore, approaches like the use of self-replicating RNA are encouraging to simplify the currently used time-consuming transfection protocols as well as to prolong the effect in patients. Especially for the directed differentiation of cells, mRNA technology is beneficial, due to the rapid protein expression and the transient persistence of IVT mRNA in the cytosol. mRNAs encoding differentiation factors can easily be delivered into the cells in a sequential and time-sensitive fashion, without the need to remove them. In addition, the amount of protein is simply controllable by adjusting the mRNA concentration. Therefore, mRNA technology is ideal for experiments that need fine-tuning of different factors. However, there are still challenges in mRNA-based applications that have to be solved (Table 3). For instance, despite modifications of the IVT mRNA molecule and the improved manufacturing technology, synthetic mRNA can still be recognized by the innate immune system. For vaccination approaches, this immune activation can be beneficial. However, for nonimmunotherapy-related applications like reprogramming of cells or protein replacement therapies, immune activation can negatively affect the therapeutic outcome, especially when repeated administration of IVT mRNA is required. Furthermore, the IVT mRNA delivery method and the vehicle for the uptake of mRNA into the cells can also induce immune responses and determine the efficiency of IVT mRNA treatment. For example, the reprogramming of cells with IVT mRNAs requires multiple daily transfections for the successful generation of iPSCs. Thus, to enable repeated transfection of cells with IVT mRNA and the continuous production of exogenous proteins, the additional use of interferon inhibitors is required. In this regard, the simultaneous use of immune inhibitors could improve the outcome of nonimmunotherapy-related IVT mRNA applications. Table 3 Advantages and disadvantages of synthetic mRNA approaches Advantages . Disadvantages . Optimization possibilities . Nonintegrative gene delivery method that minimizes the risk of insertional mutagenesis of the host genome Immunogenicity: Activation of the innate immune system Diminishing immunostimulation: Addition of immune inhibitors (e.g., B18R) Chemical modifications to mRNA (incorporation of modified nucleosides, 5′-capping) Purification of IVT mRNA (e.g., HPLC) High efficient protein expression Poor stability: Short half-life of mRNA molecules in the cytoplasm Increase of resistance against degradation: Chemical modifications of mRNA (3′-poly(A) tail) Packaging of mRNA in transport vehicles to avoid nuclease degradation (liposomes or forming of complexes with cationic polymers) Rapid, transient, and adjustable protein expression and great homogeneity among transfected cells Translation limitations Enhancement of transcriptional/translational capacity Incorporation of modified nucleosides, anti-reverse cap structure (e.g., ARCA) Modification of the promoter region (strong promoters: e.g., Kozak; IRESa, 2A peptidesb) optimization of the coding sequence: codon usage, enhancing GC content, reduction of mRNA secondary structures Cell cycle independent method (exogenous protein expression in dividing and nondividing cells) Toxicity of transfection reagents Optimization of: delivery method: chemical or physical transfection of cells the composition of transport vehicle: lipo- and polyplexes, nanoparticles, cell penetrating peptides transfection protocol for each cell type (transfection media, time course) No limitations in target size/length or number of different targets at one time (multi-target protein expression) Time consuming if repeated administration is needed (e.g., for reprogramming) Optimization of mRNA molecules and delivery method, e.g., use of: self-assembling mRNA nanoparticles self-replicating RNAs (replicons) Advantages . Disadvantages . Optimization possibilities . Nonintegrative gene delivery method that minimizes the risk of insertional mutagenesis of the host genome Immunogenicity: Activation of the innate immune system Diminishing immunostimulation: Addition of immune inhibitors (e.g., B18R) Chemical modifications to mRNA (incorporation of modified nucleosides, 5′-capping) Purification of IVT mRNA (e.g., HPLC) High efficient protein expression Poor stability: Short half-life of mRNA molecules in the cytoplasm Increase of resistance against degradation: Chemical modifications of mRNA (3′-poly(A) tail) Packaging of mRNA in transport vehicles to avoid nuclease degradation (liposomes or forming of complexes with cationic polymers) Rapid, transient, and adjustable protein expression and great homogeneity among transfected cells Translation limitations Enhancement of transcriptional/translational capacity Incorporation of modified nucleosides, anti-reverse cap structure (e.g., ARCA) Modification of the promoter region (strong promoters: e.g., Kozak; IRESa, 2A peptidesb) optimization of the coding sequence: codon usage, enhancing GC content, reduction of mRNA secondary structures Cell cycle independent method (exogenous protein expression in dividing and nondividing cells) Toxicity of transfection reagents Optimization of: delivery method: chemical or physical transfection of cells the composition of transport vehicle: lipo- and polyplexes, nanoparticles, cell penetrating peptides transfection protocol for each cell type (transfection media, time course) No limitations in target size/length or number of different targets at one time (multi-target protein expression) Time consuming if repeated administration is needed (e.g., for reprogramming) Optimization of mRNA molecules and delivery method, e.g., use of: self-assembling mRNA nanoparticles self-replicating RNAs (replicons) aIRES sequences allow the 5′ cap-independent initiation of translation of multiple genes on a single mRNA strand [111]. bThe 2A peptide sequence impairs normal peptide bond formation through a mechanism of ribosomal skipping and results in the co-translational cleavage of proteins [112]. Abbreviations: ARCA, anti-reverse cap analog; HPLC, high-performance liquid chromatography; IRES, internal ribosome entry sites; mRNA, messenger RNA; IVT, in vitro transcribed. Open in new tab Table 3 Advantages and disadvantages of synthetic mRNA approaches Advantages . Disadvantages . Optimization possibilities . Nonintegrative gene delivery method that minimizes the risk of insertional mutagenesis of the host genome Immunogenicity: Activation of the innate immune system Diminishing immunostimulation: Addition of immune inhibitors (e.g., B18R) Chemical modifications to mRNA (incorporation of modified nucleosides, 5′-capping) Purification of IVT mRNA (e.g., HPLC) High efficient protein expression Poor stability: Short half-life of mRNA molecules in the cytoplasm Increase of resistance against degradation: Chemical modifications of mRNA (3′-poly(A) tail) Packaging of mRNA in transport vehicles to avoid nuclease degradation (liposomes or forming of complexes with cationic polymers) Rapid, transient, and adjustable protein expression and great homogeneity among transfected cells Translation limitations Enhancement of transcriptional/translational capacity Incorporation of modified nucleosides, anti-reverse cap structure (e.g., ARCA) Modification of the promoter region (strong promoters: e.g., Kozak; IRESa, 2A peptidesb) optimization of the coding sequence: codon usage, enhancing GC content, reduction of mRNA secondary structures Cell cycle independent method (exogenous protein expression in dividing and nondividing cells) Toxicity of transfection reagents Optimization of: delivery method: chemical or physical transfection of cells the composition of transport vehicle: lipo- and polyplexes, nanoparticles, cell penetrating peptides transfection protocol for each cell type (transfection media, time course) No limitations in target size/length or number of different targets at one time (multi-target protein expression) Time consuming if repeated administration is needed (e.g., for reprogramming) Optimization of mRNA molecules and delivery method, e.g., use of: self-assembling mRNA nanoparticles self-replicating RNAs (replicons) Advantages . Disadvantages . Optimization possibilities . Nonintegrative gene delivery method that minimizes the risk of insertional mutagenesis of the host genome Immunogenicity: Activation of the innate immune system Diminishing immunostimulation: Addition of immune inhibitors (e.g., B18R) Chemical modifications to mRNA (incorporation of modified nucleosides, 5′-capping) Purification of IVT mRNA (e.g., HPLC) High efficient protein expression Poor stability: Short half-life of mRNA molecules in the cytoplasm Increase of resistance against degradation: Chemical modifications of mRNA (3′-poly(A) tail) Packaging of mRNA in transport vehicles to avoid nuclease degradation (liposomes or forming of complexes with cationic polymers) Rapid, transient, and adjustable protein expression and great homogeneity among transfected cells Translation limitations Enhancement of transcriptional/translational capacity Incorporation of modified nucleosides, anti-reverse cap structure (e.g., ARCA) Modification of the promoter region (strong promoters: e.g., Kozak; IRESa, 2A peptidesb) optimization of the coding sequence: codon usage, enhancing GC content, reduction of mRNA secondary structures Cell cycle independent method (exogenous protein expression in dividing and nondividing cells) Toxicity of transfection reagents Optimization of: delivery method: chemical or physical transfection of cells the composition of transport vehicle: lipo- and polyplexes, nanoparticles, cell penetrating peptides transfection protocol for each cell type (transfection media, time course) No limitations in target size/length or number of different targets at one time (multi-target protein expression) Time consuming if repeated administration is needed (e.g., for reprogramming) Optimization of mRNA molecules and delivery method, e.g., use of: self-assembling mRNA nanoparticles self-replicating RNAs (replicons) aIRES sequences allow the 5′ cap-independent initiation of translation of multiple genes on a single mRNA strand [111]. bThe 2A peptide sequence impairs normal peptide bond formation through a mechanism of ribosomal skipping and results in the co-translational cleavage of proteins [112]. Abbreviations: ARCA, anti-reverse cap analog; HPLC, high-performance liquid chromatography; IRES, internal ribosome entry sites; mRNA, messenger RNA; IVT, in vitro transcribed. Open in new tab One of the major challenges of in vivo applications is the efficient targeting of desired cells and the site-specific application of IVT mRNA to avoid systemic exposure. Therefore, new techniques and formulations are still required to improve the efficiency and safety of IVT mRNA therapeutics for in vivo applications. To date, in order to address the performance of therapeutic IVT mRNA, target protein production and the cytokine response are measured. Other critical steps in the process of mRNA-mediated protein expression, like cellular uptake, endosomal escape, translation rates, translation inhibition, and innate immune activation pathways are often not evaluated. For further optimization of IVT mRNA technology, it would be helpful to better understand these mechanisms to specialize IVT mRNA features and delivery vehicles. Transport vehicles or nanoparticles formulated with targeting residues for the recognition of specific cell types or antigens are promising to improve the effectiveness of mRNA delivery to a site-specific region. The further development of cell type-specific vehicles, which target hard-to-transfect cells such as keratinocytes, would also broaden the range of in vivo applications. We are confident that the progressive development of this technology will facilitate the translation from bench to bedside and will open the way to highly personalized and fascinating new therapies for numerous clinical applications in the future. Acknowledgments The authors would like to thank the European Social Funds in Baden-Wuerttemberg, Germany and the Ministry of Science, Research, and Art in Baden-Wuerttemberg (MWK-BW) for the financial support. Author Contributions H.S., H.P.W., and M.A.A.: conception and design; H.S., A.B., and M.A.A.: collection and assembly of data; H.S., A.B., and M.A.A.: data analysis and interpretation; H.S. and M.A.A.: manuscript writing; C.S. and H.P.W.: administrative support; H.S., C.S., H.P.W., and M.A.A.: final approval of manuscript. Disclosure The author(s) indicates no potential conflicts of interest. References 1 Griesenbach U , Pytel KM Alton EW. Cystic fibrosis gene therapy in the UK and Elsewhere . Hum Gene Ther 2015 ; 26 : 266 – 275 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Bangel-Ruland N , Tomczak K, Fernandez Fernandez E et al. Cystic fibrosis transmembrane conductance regulator-mRNA delivery: A novel alternative for cystic fibrosis gene therapy . J Gene Med 2013 ; 15 : 414 – 426 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Kormann MS , Hasenpusch G, Aneja MK et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice . Nat Biotechnol 2011 ; 29 : 154 – 157 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Garcia F , Plana M, Climent N et al. Dendritic cell based vaccines for HIV infection: The way ahead . Hum Vaccin Immunother 2013 ; 9 : 2445 – 2452 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Petsch B , Schnee M, Vogel AB et al. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza a virus infection . Nat Biotechnol 2012 ; 30 : 1210 – 1216 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Benteyn D , Anguille S, Van Lint S et al. Design of an optimized Wilms' tumor 1 (WT1) mRNA construct for enhanced WT1 expression and improved immunogenicity in vitro and in vivo . Mol Ther Nucleic Acids 2013 ; 2 : e134 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Levy O , Zhao W, Mortensen LJ et al. mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation . Blood 2013 ; 122 : e23 – e32 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Takahashi K Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors . Cell 2006 ; 126 : 663 – 676 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Warren L , Manos PD, Ahfeldt T et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA . Cell Stem Cell 2010 ; 7 : 618 – 630 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Stadtfeld M , Nagaya M, Utikal J et al. Induced pluripotent stem cells generated without viral integration . Science 2008 ; 322 : 945 – 949 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Woltjen K , Michael IP, Mohseni P et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells . Nature 2009 ; 458 : 766 – 770 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Yu J , Hu K, Smuga-Otto K et al. Human induced pluripotent stem cells free of vector and transgene sequences . Science 2009 ; 324 : 797 – 801 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Seki T , Yuasa S, Oda M et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells . Cell Stem Cell 2010 ; 7 : 11 – 14 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Yoshioka N , Gros E, Li HR et al. Efficient generation of human iPSCs by a synthetic self-replicative RNA . Cell Stem Cell 2013 ; 13 : 246 – 254 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Rao MS , Malik N. Assessing iPSC reprogramming methods for their suitability in translational Medicine . J Cell Biochem 2012 ; 113 : 3061 – 3068 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Steichen C , Luce E, Maluenda J et al. Messenger RNA versus retrovirus-based induced pluripotent stem cell reprogramming strategies: Analysis of genomic integrity . Stem Cells Transl Med 2014 ; 3 : 686 – 691 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Mallon BS , Hamilton RS, Kozhich OA et al. Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin . Stem Cell Res 2014 ; 12 : 376 – 386 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Okita K , Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells . Nature 2007 ; 448 : 313 – 317 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Zhao T , Zhang ZN, Rong Z et al. Immunogenicity of induced pluripotent stem cells . Nature 2011 ; 474 : 212 – 215 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Hu K. All roads lead to induced pluripotent stem cells: The technologies of iPSC generation . Stem Cells Dev 2014 ; 23 : 1285 – 1300 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Kaji K , Norrby K, Paca A et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors . Nature 2009 ; 458 : 771 – 775 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Yakubov E , Rechavi G, Rozenblatt S et al. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors . Biochem Biophys Res Commun 2010 ; 394 : 189 – 193 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Plews JR , Li J, Jones M et al. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach . PLoS One 2010 ; 5 : e14397 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Schlaeger TM , Daheron L, Brickler TR et al. A comparison of non-integrating reprogramming methods . Nat Biotechnol 2015 ; 33 : 58 – 63 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Zangi L , Lui KO, von Gise A et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction . Nat Biotechnol 2013 ; 31 : 898 – 907 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Rettig L , Haen SP, Bittermann AG et al. Particle size and activation threshold: A new dimension of danger signaling . Blood 2010 ; 115 : 4533 – 4541 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Hu K. Vectorology and factor delivery in induced pluripotent stem cell reprogramming . Stem Cells Dev 2014 ; 23 : 1301 – 1315 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Bernal JA. RNA-based tools for nuclear reprogramming and lineage-conversion: Towards clinical applications . J Cardiovasc Transl Res 2013 ; 6 : 956 – 968 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Sahin U , Kariko K, Tureci O. mRNA-based therapeutics–Developing a new class of drugs . Nat Rev Drug Discov 2014 ; 13 : 759 – 780 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Kariko K , Muramatsu H, Ludwig J et al. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA . Nucleic Acids Res 2011 ; 39 : e142 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Kariko K , Muramatsu H, Welsh FA et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability . Mol Ther 2008 ; 16 : 1833 – 1840 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Loomis KH , Kirschman JL, Bhosle S et al. Strategies for modulating innate immune activation and protein production of in vitro transcribed mRNAs . J Mater Chem B 2016 ; 4 : 1619 – 1632 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Hawley-Nelson P , Ciccarone V, Moore ML. Transfection of cultured eukaryotic cells using cationic lipid reagents . Curr Protoc Mol Biol 2008 ;chapter 9: Unit 9 4. Google Scholar OpenURL Placeholder Text WorldCat 34 Lorenz C , Fotin-Mleczek M, Roth G et al. Protein expression from exogenous mRNA: Uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway . RNA Biol 2011 ; 8 : 627 – 636 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Bire S , Gosset D, Jegot G et al. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition . BMC Biotechnol 2013 ; 13 : 75 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Van Tendeloo VF , Ponsaerts P, Berneman ZN. mRNA-based gene transfer as a tool for gene and cell therapy . Curr Opin Mol Ther 2007 ; 9 : 423 – 431 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 37 Alexopoulou L , Holt AC, Medzhitov R et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3 . Nature 2001 ; 413 : 732 – 738 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Kariko K , Ni H, Capodici J et al. mRNA is an endogenous ligand for Toll-like receptor 3 . J Biol Chem 2004 ; 279 : 12542 – 12550 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Diebold SS , Kaisho T, Hemmi H et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA . Science 2004 ; 303 : 1529 – 1531 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Diebold SS , Massacrier C, Akira S et al. Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides . Eur J Immunol 2006 ; 36 : 3256 – 3267 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Heil F , Hemmi H, Hochrein H et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8 . Science 2004 ; 303 : 1526 – 1529 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Lorenzi JC , Trombone AP, Rocha CD et al. Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis . BMC Biotechnol 2010 ; 10 : 77 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Schlee M , Roth A, Hornung V et al. Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus . Immunity 2009 ; 31 : 25 – 34 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Schmidt A , Schwerd T, Hamm W et al. 5′-Triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I . Proc Natl Acad Sci U S A 2009 ; 106 : 12067 – 12072 . Google Scholar Crossref Search ADS PubMed WorldCat 45 Goubau D , Schlee M, Deddouche S et al. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates . Nature 2014 ; 514 : 372 – 375 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Devarkar SC , Wang C, Miller MT et al. Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I . Proc Natl Acad Sci U S A 2016 ; 113 : 596 – 601 . Google Scholar Crossref Search ADS PubMed WorldCat 47 Schuberth-Wagner C , Ludwig J, Bruder AK et al. A conserved histidine in the RNA sensor RIG-I controls immune tolerance to N1-2′O-methylated self RNA . Immunity 2015 ; 43 : 41 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 48 Kato H , Takeuchi O, Mikamo-Satoh E et al. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5 . J Exp Med 2008 ; 205 : 1601 – 1610 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Zust R , Cervantes-Barragan L, Habjan M et al. Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5 . Nat Immunol 2011 ; 12 : 137 – 143 . Google Scholar Crossref Search ADS PubMed WorldCat 50 Pichlmair A , Schulz O, Tan CP et al. Activation of MDA5 requires higher-order RNA structures generated during virus infection . J Virol 2009 ; 83 : 10761 – 10769 . Google Scholar Crossref Search ADS PubMed WorldCat 51 Kanneganti TD , Body-Malapel M, Amer A et al. Critical role for cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA . J Biol Chem 2006 ; 281 : 36560 – 36568 . Google Scholar Crossref Search ADS PubMed WorldCat 52 Sabbah A , Chang TH, Harnack R et al. Activation of innate immune antiviral responses by Nod2 . Nat Immunol 2009 ; 10 : 1073 – 1080 . Google Scholar Crossref Search ADS PubMed WorldCat 53 Devoldere J , Dewitte H, De Smedt SC et al. Evading innate immunity in nonviral mRNA delivery: Don't shoot the messenger . Drug Discov Today 2016 ; 21 : 11 – 25 . Google Scholar Crossref Search ADS PubMed WorldCat 54 Loo YM , Gale M Jr., Immune signaling by RIG-I-like receptors . Immunity 2011 ; 34 : 680 – 692 . Google Scholar Crossref Search ADS PubMed WorldCat 55 Andries O , De Filette M, De Smedt SC et al. Innate immune response and programmed cell death following carrier-mediated delivery of unmodified mRNA to respiratory cells . J Control Release 2013 ; 167 : 157 – 166 . Google Scholar Crossref Search ADS PubMed WorldCat 56 Nallagatla SR , Bevilacqua PC. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner . RNA 2008 ; 14 : 1201 – 1213 . Google Scholar Crossref Search ADS PubMed WorldCat 57 Angel M , Yanik MF. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins . PLoS One 2010 ; 5 : e11756 . Google Scholar Crossref Search ADS PubMed WorldCat 58 Hoerr I , Obst R, Rammensee HG et al. In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies . Eur J Immunol 2000 ; 30 : 1 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 59 Kariko K , Buckstein M, Ni H et al. Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA . Immunity 2005 ; 23 : 165 – 175 . Google Scholar Crossref Search ADS PubMed WorldCat 60 Kallen KJ , Thess A. A development that may evolve into a revolution in medicine: mRNA as the basis for novel, nucleotide-based vaccines and drugs . Ther Adv Vaccines 2014 ; 2 : 10 – 31 . Google Scholar Crossref Search ADS PubMed WorldCat 61 Kariko K , Weissman D. Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implication for therapeutic RNA development . Curr Opin Drug Discov Dev 2007 ; 10 : 523 – 532 . Google Scholar OpenURL Placeholder Text WorldCat 62 Quabius ES , Krupp G. Synthetic mRNAs for manipulating cellular phenotypes: An overview . N Biotechnol 2015 ; 32 : 229 – 235 . Google Scholar Crossref Search ADS PubMed WorldCat 63 Mahiny AJ , Dewerth A, Mays LE et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency . Nat Biotechnol 2015 ; 33 : 584 – 586 . Google Scholar Crossref Search ADS PubMed WorldCat 64 Poleganov MA , Eminli S, Beissert T et al. Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using nonmodified RNA for reprogramming and immune evasion . Hum Gene Ther 2015 ; 26 : 751 – 766 . Google Scholar Crossref Search ADS PubMed WorldCat 65 Preskey D , Allison TF, Jones M et al. Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts . Biochem Biophys Res Commun 2015 ; 3 : 743 – 751 . Google Scholar OpenURL Placeholder Text WorldCat 66 Wang Y , Su HH, Yang Y et al. Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy . Mol Ther 2013 ; 21 : 358 – 367 . Google Scholar Crossref Search ADS PubMed WorldCat 67 Kariko K , Muramatsu H, Keller JM et al. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin . Mol Ther 2012 ; 20 : 948 – 953 . Google Scholar Crossref Search ADS PubMed WorldCat 68 Andries O , Mc Cafferty S, De Smedt SC et al. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice . J Control Release 2015 ; 217 : 337 – 344 . Google Scholar Crossref Search ADS PubMed WorldCat 69 Pardi N , Tuyishime S, Muramatsu H et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes . J Control Release 2015 ; 217 : 345 – 351 . Google Scholar Crossref Search ADS PubMed WorldCat 70 Mays LE , Ammon-Treiber S, Mothes B et al. Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism . J Clin Invest 2013 ; 123 : 1216 – 1228 . Google Scholar Crossref Search ADS PubMed WorldCat 71 Malone RW , Felgner PL, Verma IM. Cationic liposome-mediated RNA transfection . Proc Natl Acad Sci U S A 1989 ; 86 : 6077 – 6081 . Google Scholar Crossref Search ADS PubMed WorldCat 72 Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs . Nucleic Acids Res 1987 ; 15 : 8125 – 8148 . Google Scholar Crossref Search ADS PubMed WorldCat 73 Mockey M , Goncalves C, Dupuy FP et al. mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with poly(a) chains in cis and in trans for a high protein expression level . Biochem Biophys Res Commun 2006 ; 340 : 1062 – 1068 . Google Scholar Crossref Search ADS PubMed WorldCat 74 Grudzien-Nogalska E , Kowalska J, Su W et al. Synthetic mRNAs with superior translation and stability properties . Methods Mol Biol 2013 ; 969 : 55 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 75 Stepinski J , Waddell C, Stolarski R et al. Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl (3′-deoxy)GpppG . RNA 2001 ; 7 : 1486 – 1495 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 76 Grudzien-Nogalska E , Jemielity J, Kowalska J et al. Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells . RNA 2007 ; 13 : 1745 – 1755 . Google Scholar Crossref Search ADS PubMed WorldCat 77 Hornung V , Ellegast J, Kim S et al. 5′-Triphosphate RNA is the ligand for RIG-I . Science 2006 ; 314 : 994 – 997 . Google Scholar Crossref Search ADS PubMed WorldCat 78 Pichlmair A , Schulz O, Tan CP et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates . Science 2006 ; 314 : 997 – 1001 . Google Scholar Crossref Search ADS PubMed WorldCat 79 Goldstrohm AC , Wickens M. Multifunctional deadenylase complexes diversify mRNA control . Nat Rev Mol Cell Biol 2008 ; 9 : 337 – 344 . Google Scholar Crossref Search ADS PubMed WorldCat 80 Drews K , Tavernier G, Demeester J et al. The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts . Biomaterials 2012 ; 33 : 4059 – 4068 . Google Scholar Crossref Search ADS PubMed WorldCat 81 Rautsi O , Lehmusvaara S, Salonen T et al. Type I interferon response against viral and non-viral gene transfer in human tumor and primary cell lines . J Gene Med 2007 ; 9 : 122 – 135 . Google Scholar Crossref Search ADS PubMed WorldCat 82 Avci-Adali M , Behring A, Keller T et al. Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression . J Biol Eng 2014 ; 8 : 8 . Google Scholar Crossref Search ADS PubMed WorldCat 83 Schwartz SD , Hubschman JP, Heilwell G et al. Embryonic stem cell trials for macular degeneration: A preliminary report . Lancet 2012 ; 379 : 713 – 720 . Google Scholar Crossref Search ADS PubMed WorldCat 84 Chen Z , Zhang YA. Cell therapy for macular degeneration–First phase I/II pluripotent stem cell-based clinical trial shows promise . Sci China Life Sci 2015 ; 58 : 119 – 120 . Google Scholar Crossref Search ADS PubMed WorldCat 85 Reardon S , Cyranoski D. Japan stem-cell trial stirs envy . Nature 2014 ; 513 : 287 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat 86 Kanemura H , Go MJ, Shikamura M et al. Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration . PLoS One 2014 ; 9 : e85336 . Google Scholar Crossref Search ADS PubMed WorldCat 87 Cyranoski D. Japanese woman is first recipient of next-generation stem cells . Available at http://www.nature.com/news/japanese-woman-is-first-recipient-of-next-generation-stem-cells-1.15915. Accessed January 16 , 2016 . 88 Lee AS , Tang C, Rao MS et al. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies . Nat Med 2013 ; 19 : 998 – 1004 . Google Scholar Crossref Search ADS PubMed WorldCat 89 Huangfu D , Osafune K, Maehr R et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2 . Nat Biotechnol 2008 ; 26 : 1269 – 1275 . Google Scholar Crossref Search ADS PubMed WorldCat 90 Hirai H , Tani T, Katoku-Kikyo N et al. Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD . Stem Cells 2011 ; 29 : 1349 – 1361 . Google Scholar Crossref Search ADS PubMed WorldCat 91 Warren L , Ni Y, Wang J et al. Feeder-free derivation of human induced pluripotent stem cells with messenger RNA . Sci Rep 2012 ; 2 : 657 . Google Scholar Crossref Search ADS PubMed WorldCat 92 Liu X , Sun H, Qi J et al. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming . Nat Cell Biol 2013 ; 15 : 829 – 838 . Google Scholar Crossref Search ADS PubMed WorldCat 93 Sancho-Martinez I , Baek SH, Izpisua Belmonte JC. Lineage conversion methodologies meet the reprogramming toolbox . Nat Cell Biol 2012 ; 14 : 892 – 899 . Google Scholar Crossref Search ADS PubMed WorldCat 94 Muraoka N , Ieda M. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis . Annu Rev Physiol 2014 ; 76 : 21 – 37 . Google Scholar Crossref Search ADS PubMed WorldCat 95 Qian L , Huang Y, Spencer CI et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes . Nature 2012 ; 485 : 593 – 598 . Google Scholar Crossref Search ADS PubMed WorldCat 96 Szabo E , Rampalli S, Risueno RM et al. Direct conversion of human fibroblasts to multilineage blood progenitors . Nature 2010 ; 468 : 521 – 526 . Google Scholar Crossref Search ADS PubMed WorldCat 97 Zhou Q , Brown J, Kanarek A et al. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells . Nature 2008 ; 455 : 627 – 632 . Google Scholar Crossref Search ADS PubMed WorldCat 98 Sekiya S , Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors . Nature 2011 ; 475 : 390 – 393 . Google Scholar Crossref Search ADS PubMed WorldCat 99 Simeonov KP , Uppal H. Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs . PLoS One 2014 ; 9 : e100134 . Google Scholar Crossref Search ADS PubMed WorldCat 100 Vierbuchen T , Ostermeier A, Pang ZP et al. Direct conversion of fibroblasts to functional neurons by defined factors . Nature 2010 ; 463 : 1035 – 1041 . Google Scholar Crossref Search ADS PubMed WorldCat 101 Kim H , Park Y, Lee JB. Self-assembled messenger RNA nanoparticles (mRNA-NPs) for efficient gene expression . Sci Rep 2015 ; 5 : 12737 . Google Scholar Crossref Search ADS PubMed WorldCat 102 Sadelain M , Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design . Cancer Discov 2013 ; 3 : 388 – 398 . Google Scholar Crossref Search ADS PubMed WorldCat 103 Bringmann A , Held SA, Heine A et al. RNA vaccines in cancer treatment . J Biomed Biotechnol 2010 ;2010 : 623687 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 104 Barrett DM , Zhao Y, Liu X et al. Treatment of advanced leukemia in mice with mRNA engineered T cells . Hum Gene Ther 2011 ; 22 : 1575 – 1586 . Google Scholar Crossref Search ADS PubMed WorldCat 105 Beatty GL , Haas AR, Maus MV et al. Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies . Cancer Immunol Res 2014 ; 2 : 112 – 120 . Google Scholar Crossref Search ADS PubMed WorldCat 106 Kenderian SS , Ruella M, Shestova O et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia . Leukemia 2015 ; 29 : 1637 – 1647 . Google Scholar Crossref Search ADS PubMed WorldCat 107 Michel T , Kankura A, Salinas Medina ML et al. In vitro evaluation of a novel mRNA-based therapeutic strategy for the treatment of patients suffering from alpha-1-antitrypsin deficiency . Nucleic Acid Ther 2015 ; 25 : 235 – 244 . Google Scholar Crossref Search ADS PubMed WorldCat 108 Schlake T , Thess A, Fotin-Mleczek M et al. Developing mRNA-vaccine technologies . RNA Biol 2012 ; 9 : 1319 – 1330 . Google Scholar Crossref Search ADS PubMed WorldCat 109 Rausch S , Schwentner C, Stenzl A et al. mRNA vaccine CV9103 and CV9104 for the treatment of prostate cancer . Hum Vaccin Immunother 2014 ; 10 : 3146 – 3152 . Google Scholar Crossref Search ADS PubMed WorldCat 110 Sebastian M , Papachristofilou A, Weiss C et al. Phase Ib study evaluating a self-adjuvanted mRNA cancer vaccine (RNActive(R)) combined with local radiation as consolidation and maintenance treatment for patients with stage IV non-small cell lung cancer . BMC Cancer 2014 ; 14 : 748 . Google Scholar Crossref Search ADS PubMed WorldCat 111 Hellen CU , Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules . Genes Dev 2001 ; 15 : 1593 – 1612 . Google Scholar Crossref Search ADS PubMed WorldCat 112 Trichas G , Begbie J, Srinivas S. Use of the viral 2A peptide for bicistronic expression in transgenic mice . BMC Biol 2008 ; 6 : 40 – 40 . Google Scholar Crossref Search ADS PubMed WorldCat © 2016 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges
JF - Stem Cells
DO - 10.1002/stem.2402
DA - 2017-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/concise-review-application-of-in-vitro-transcribed-messenger-rna-for-JbM2U8S55p
SP - 68
EP - 79
VL - 35
IS - 1
DP - DeepDyve
ER -