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Improved translation efficiency of therapeutic mRNA
Gene 707 (2019) 231–238
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Improved translation efficiency of therapeutic mRNA a
Farzaneh Zarghampoor , Negar Azarpira Ali Mohammad Foroughmanda a b c
b,⁎
a
T c
, Saeed Reza Khatami , Abbas Behzad-Behbahani ,
Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: UTR mRNA In vitro transcription Kozak sequence Beta-globin mRNA translation Green fluorescent protein
Recent developments in the field of the messenger RNA and its advantages versus DNA have led to a renewed interest in mRNA-based technologies. Despite its advantages, mRNA therapy has a number of drawbacks including low amount of mRNA production, short-term existence of mRNA and mRNA-mediated protein within the cell, severe mRNA cytotoxicity, and immune response activation following mRNA transfection. Here, we applied untranslated regions of human beta-globin to increase the stability and translation efficiency of a destabilized GFP mRNA. In order to suppress the innate immune response, which is the main barrier of mRNA therapy, we used the vaccinia virus derived capping enzyme and substituted standard nucleotides with modified nucleotides. At the end, the Kozak sequence of human beta-globin was replaced with the strongest sequence for the further improvement of mRNA translation. Overall, these modifications with native Kozak (K1) sequence of human beta-globin enhanced the stability of destabilized GFP mRNA up to 48 h and no increase in the level of interferon-α and -β was found. The GFP expression of mRNA with modified Kozak (K2) sequence initiated earlier than mRNA and plasmid DNA with K1 sequence. In contrast to mRNA with K1 sequence, the cells containing mRNA with K2 sequence remained positive for GFP expression up to 72 h post-transfection. Interestingly, transfection efficiency and mean fluorescence intensity (MFI) of mRNA with K2 sequence were higher than mRNA and plasmid DNA with K1 sequence. Taken together, these results provide valuable information for the optimization of mRNA stability and translation. Therefore, the methods used in the current study can successfully be applied for reprogramming, gene editing, trans-differentiation, tumour therapy, and gene therapy.
1. Background Nucleic acid-based therapy was first reported by Wolff et al. (1990). At that time, gene therapy was based on viral vectors and plasmid DNA technology. Recent advances in new treatment based on mRNA and its advantages versus DNA-based therapy make mRNA an attractive platform for gene therapy. Unlike DNA-based therapies, mRNA does not need to enter the nucleus; thus, for both non-dividing and slowly-dividing cells, mRNA therapy is more effective than DNA therapy (Zou et al., 2010). In addition, mRNA does not integrate into the host genome and there is no risk of insertional mutagenesis (Bernal, 2013; Hacein-Bey-Abina et al., 2003). Transient expression is another potential usefulness of mRNA therapy, which is important for the stability of the cells. However, mRNA-based therapy has several major problems: the short half-life of mRNA and mRNA-mediated protein, low level of in vitro transcription (IVT), severe mRNA cytotoxicity, and immune
⁎
response activation following mRNA transfection (Lu and Li, 2013). Mature mRNA in the eukaryotic cells contained several major regions that influence the degradation by exonucleases and translation efficiency (Leppek et al., 2018). Of those, 5′ and 3′ untranslated regions (UTRs) are known to play crucial roles in the regulation of translation (Van Der Velden and Thomas, 1999) and stability (Bashirullah et al., 2001). Therefore, one strategy that enhances translation efficiency and mRNA stability is using the variants of UTRs. To increase the stability of mRNA, 3′UTR of various endogenous mRNAs which have large amounts of protein production and long half-life such as α-globin, albumin and, β-globin has been studied extensively (Volloch and Housman, 1981; Ross and Sullivan, 1985). Thus, the use of 3′UTR represents a logical method to increase mRNA stability. Translation efficiency could potentially be influenced by the presence of the cap structure, 5′UTR sequence, and the Kozak sequence. In the eukaryotic cells, typically pre-initiation complex of translation is
Corresponding author. E-mail address: [email protected] (N. Azarpira).
https://doi.org/10.1016/j.gene.2019.05.008 Received 1 February 2019; Received in revised form 2 May 2019; Accepted 3 May 2019 Available online 04 May 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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transfection efficiency in HEK293T cells was higher than HFF cells 24 h post-transfection (Fig. 3; i 24 h and c). To improve IVT mRNA transfection of HFF cells, we trypsinized the cells and then transfection was done in suspension. The results showed that the efficiency of modified transfection was improved compared with the conventional method (Fig. 3; c and d). The GFP expression of IVT mRNA2 (with K2 sequence) in HEK293T cells was initiated 2-h post-transfections. The cells with the IVT mRNA1 (with K1 sequence) initiated GFP expression 6-h after transfection. In contrast to IVT mRNA2, the intensity of GFP expression of IVT mRNA1 was very low (Fig. 3; i 24 h and h). Overall, the transfection rate of IVT mRNAs was higher than plasmid DNA 24 h after incubation (Fig. 3; h vs f and i 24 h vs g). The intensity of GFP expression from IVT mRNA2 was decreased approximately 36 h after transfection (Fig. 3; i 36 h). However, the HEK293T cells were positive for GFP expression up to 72 h. In comparison with IVT mRNA2, the intensity of GFP expression from IVT mRNA1 was decreased approximately 24 h after transfection. The same results were obtained when the HFF cells were transfected with the constructs.
formed near of a cap structure and then scanning of the 5′UTR reaches the start codon. Thus, the 5′UTR plays an important role in the translation efficiency as it represents protein binding sites for a pre-initiation complex of translation and secondary structures that impact on scanning the 5′UTR (Sonenberg and Hinnebusch, 2009). Recent evidence suggests that the 5′UTR is the primary driver for protein translation from IVT mRNA and modification of the 5′UTR appears to have the largest impact on translation (Asrani et al., 2018). In the vertebrates, the most efficient Kozak sequence is gccRccAUGG (R = purine). The purine R is located in position −3 and G in position +4 have the strongest effect (Kozak, 1986; Babendure et al., 2006). Perhaps, the most serious disadvantage of mRNA-based technologies is the innate immune response. Exogenous RNAs with the signature of viral nucleic acids such as uncapped 5′ triphosphate, complex secondary structures in RNA such as pseudoknots, and GU-rich RNA are pathogen-associated molecular patterns for Toll-like receptors (Wu et al., 2013; Forsbach et al., 2008; Nallagatla et al., 2007). Therefore, IVT mRNAs with this signature may interact with pattern recognition receptors (PRRs), such as TLRs (Alexopoulou et al., 2001a; Karikó et al., 2004), RIG I, MDA5, PKR, OAS-L and RLR, and induce an innate immune response (Yoneyama et al., 2004; Yoneyama et al., 2005; Balachandran et al., 2000; Donovan et al., 2013). Stimulating the innate immune response can cause a variety of effects including cell cycle arrest, translation block, upregulation of PRRs, and apoptosis (Alexopoulou et al., 2001b). To improve the stability of IVT mRNA and reduce the innate immune response, we used 5′ and 3′UTR of human beta-globin, Vaccinia virus derived capping enzyme for generation cap 1 structure, substituted cytidine and uridine with 5-methylcytidine and pseudouridine, and performed polyadenylation in the 3′ end of IVT mRNA. In addition, for enhancing protein production of IVT mRNA, we replaced the Kozak sequence of human beta-globin with the strongest Kozak sequence. Finally, the translation efficiency of both constructs with K1 and K2 sequence was evaluated in human foreskin fibroblast (HFF) cell and HEK293T cell line.
2.4. Flow cytometry assay The transfection efficiency and GFP expression intensity of plasmid DNAs and IVT mRNAs in the HFF and HEK293T cells were evaluated by flow cytometry assay 24 h post-incubation. Overall, the transfection efficiency of IVT mRNAs was higher than plasmid DNAs; however, the mean fluorescence intensity of each plasmid DNA was higher than the related IVT mRNA. In comparison with IVT mRNA1, transfection efficiency and the mean fluorescence intensity of IVT mRNA2 were higher. In addition, the stability and GFP expression intensity of both IVT mRNAs were monitored in the HEK293T cells 24, 36, 48, and 72 h after transfection. Maximal GFP expression was determined between 24 and 48 h after transfection. The percentage of GFP positive cells containing IVT mRNA2 remained stable 24 to 72 h and decreased 72 h after transfection. In contrast, the percentage of GFP positive cells containing IVT mRNA1 decreased 24 h post-transfection (Figs. 4 and 5).
2. Results 2.5. Cytotoxicity assay of plasmid DNAs, IVT mRNAs, and transfection reagents
2.1. Construction of recombinant plasmids As illustrated in Fig. 1, 5′UTR of human beta-globin (185 bp) with K1 sequence (ACAGACACCATG), 3′UTR of human beta-globin (156 bp) and destabilized GFP (GFPd2, 853 bp) were amplified by simple PCR. Following this, the DNA fragments were ligated together by SOEing PCR [5′UTR-K1-GFPd2–3′UTR]. Finally, the construct with K2 sequence (GCCGCCACCATG) was generated using the new primers for 3′ end of 5′UTR and 5′ end of GFPd2 (Fig. 1).
The effect of Lipofectamine 3000 reagent, plasmid DNAs, and IVT mRNAs on the cell viability were evaluated using MTT assay. As shown in Fig. 6, plasmid DNA tends to cause greater cell toxicity when paired with Lipofectamine 3000. However, there were no statistically significant differences between the viability of transfected and nontransfected cells after 24 h (Fig. 6). 2.6. Immune response stimulation following IVT mRNA transfection
2.2. mRNA in vitro transcription To determine the innate immune response following IVT mRNA transfection using Lipofectamine 3000, we measured the level of human interferon alpha and beta (hIFN-α/β) in the supernatant of HEK293T cells by ELISA. The concentration of hIFN-β (75.181 pg/ml) was detected some higher than hIFN-α (58.239 pg/ml). However, in comparison with non-transfected cells, no significant differences were observed in the levels of hIFN-α and -β in the supernatant of the transfected cells (Fig. 7).
For in vitro transcription, linearized plasmid DNA and the PCR product were used. Interestingly, the efficiency of in vitro transcription from a PCR product (65 μg RNA from 1 μg DNA) was higher than linearized plasmid DNA (8 μg RNA from 1 μg DNA). Furthermore, IVT mRNA produced from linearized plasmid DNA did not have any GFP expression in the HEK293T cell line. Hence, for in vitro transcription, we used PCR product as template. The quality and size of mRNAs were assessed on agarose gel electrophoresis (Fig. 2). In addition, the presence of a poly-A tail in the 3′end of IVT mRNA was confirmed with agarose gel electrophoresis.
3. Discussion IVT mRNA is a multifunctional molecule that can be applied for reprogramming, gene editing, transdifferentiation, tumour therapy, vaccination, and gene therapy. In recent years, researchers have shown an increased interest in IVT mRNA due to important differences between IVT mRNA and plasmid DNA. Compared with plasmid DNA, IVT mRNA has a transient nature; therefore, it probably offers improved
2.3. IVT mRNA and plasmid DNA transfection To assess the protein expression efficiency of K1 and K2 sequences, we transfected IVT mRNA and the plasmid DNA in the HFF and HEK293T cells. As shown in Fig. 3, using fluorescent microscopy, the 232
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Fig. 1. Construction of recombinant plasmids. (a) 5′UTR of human beta-globin containing K1 and K2 sequence was amplified using simple PCR. The 3′UTR of betaglobin and GFPd2 were amplified by simple PCR, as well. (b) The DNA fragments were spliced together, using SOEing PCR. (c) Subsequently, both of the constructs with K1 and K2 sequences were inserted into pcDNA3.1+ vector by calcium chloride method. (d) Agarose gel electrophoresis of simple and SOEing PCRs; (1) 100 bp DNA ladder, (2 and 3) 5′UTR of human beta-globin with K1 and K2 sequence (185 bp), (4) 3′UTR of human beta-globin (156 bp), (5) GFPd2 fragment (853 bp), (6) the SOEing PCR1 product (5′UTR-GFPd2, 1011 bp), and (7) the SOEing PCR2 product (5′UTR-GFPd2–3′UTR, 1147 bp). K1: native Kozak sequence; K2: modified Kozak sequence; Construct1: 5′UTR-K1-GFPd2–3′UTR; Construct2: 5′UTR- K2-GFPd2–3′UTR.
complex. Additionally, IVT mRNA is often delivered into the cell through the endolysosomal process. Therefore, the presence of abnormal IVT mRNA in the extracellular, endosomal, and cytoplasmic compartment, particularly the 5′ triphosphate mRNA, led to its detection by PRRs (such as PKR and RIGI). The PKR pathway is the most harmful antiviral pathway that leads to inhibition of protein expression from endogenous and exogenous mRNA. Activation of PRRs leads to the initiation of a cascade signalling that results in up-regulation of type I interferon and NF-kB related genes. On the other hand, Translation in the eukaryotic cells almost begins in the cap-dependent mechanism. Consequently, a key step in processing IVT mRNA is the addition of a cap structure into the 5′ end of mRNA. There are different capping systems to incorporate the cap structure in the 5′ end of IVT mRNA. To suppress the innate immune response, Warren et al. used cap analog ARCA and substituted modified nucleotides with the unmodified counterpart (substitution of cytidine and uridine with 5-methylcytidine and pseudouridine) (Warren et al., 2010). However, still the residual up-regulation of some targets of the interferon on the gene expression level was detected. Other studies have shown that the substitution of uridine with pseudouridine, suppresses the innate immune response and increases the protein expression (Anderson et al., 2010). In the present study, to suppress the immune response and increase translation, we used vaccinia virus derived capping enzyme to incorporate a cap1 structure into the 5′ end of IVT mRNA. The efficiency of this capping system is nearly 100% whereas efficiency cap analog ARCA is ~80% and cap analog is ~50% of IVT, and need RNA products purified or treated with phosphatase to eliminate any remaining 5′triphosphates. Additionally, we substituted cytidine and uridine with 5-methylcytidine and pseudouridine,
control over protein expression kinetics and nucleic acid dosing. For some cell types, such as non-dividing or slowly-dividing cells, IVT mRNA leads to more effective protein production (Zou et al., 2010; Wang et al., 2013). Furthermore, IVT mRNA remains in the cytoplasm and does not enter the nucleus; hence, there is no risk of integration into the host genome (Bernal, 2013; Hacein-Bey-Abina et al., 2003). Despite its safety and efficacy, IVT mRNA has several major drawbacks which should be overcome. One of the problems of IVT mRNA is the deficiency for scalable production of in vitro transcribed RNA. As previous studies have shown, the chemical in vitro RNA synthesis is inappropriate for large production of long templates (> 20nts) (Lu and Li, 2013; Nelissen et al., 2012). Therefore, in the present study for the large production of IVT mRNA using T7 RNA polymerase, linearized plasmid or PCR amplicon was used as the template. In comparison with the linearized plasmid, higher yield of mRNA was synthesized when the PCR amplified DNA was used as a template for in vitro transcription. The same scale of in vitro transcription has been reported when the PCR amplicon was used as a template for the large scale production of mRNA (Rohani et al., 2016). Although the reason has not been clearly described, the quality and purity of linearized plasmid may be the main reason for the low efficiency of in vitro transcription. Degradation of linearized plasmid or contamination with RNase during purification may have an adverse effect on the yield of in vitro transcription. Due to the large size of plasmid DNA molecules, it could be possible to damage the linearized plasmid DNA during purification in comparison with PCR amplicon. The main limitations of IVT mRNA for therapeutic applications are low stability and innate immune stimulation. Unlike IVT mRNA, endogenous mRNA is almost entirely in RNPs (Ribonucleo proteins) 233
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other modifications increased the stability and translation of GFPd2 to 48 h after transfection. However, the percentage of positive cells containing IVT mRNA with K2 sequence remained stable up to 72 h after transfection. On the other hands, the transfected cells with IVT mRNA2 initiated GFP expression earlier than IVT mRNA1. It is interesting to note that the amount of protein expression from each plasmid DNA was higher than IVT mRNA respective. However, the amount of protein expression from IVT mRNA2 was higher than plasmid DNA and IVT mRNA1. These results were not due to the higher stability of IVT mRNA2 than IVT mRNA1, in fact, the performance of modified Kozk sequence and enhancing in protein translation efficiency are the two main reasons. As previously mentioned, the translation of mRNA is a complex process involving multiple protein-protein and protein-mRNA interactions. These RNA binding proteins bind to the endogenous mRNA prior to export from the nucleus and can impact protein production (Wickramasinghe and Laskey, 2015). While IVT mRNA directly is delivered to the cytosol and would bypass these traditional modifications. This may explain the differences in time of initiation of GFP expression and relative protein expression from plasmid DNA and IVT mRNA. 4. Conclusions In summary, the current study provides important information to increase the stability and efficiency of mRNA translation, to suppress the immune response that main of hurdles of IVT mRNA, and highlight the main role of 5′UTR in enhancing protein production. In addition, using the modified Kozak sequence in the human 5′UTR beta-globin had a positive impact on the translation efficiency of IVT mRNA which is the new finding of this study.
Fig. 2. Agarose gel electrophoresis of heat-denatured IVT mRNA products. (1) Pseudouridine RNA Molecular Weight Markers, (2) IVT mRNA without cap and poly-A tail, (3): IVT mRNA with cap1 and poly-A tail.
5. Materials and methods
respectively. Hence, in supernatant transfected cells no increases were detected in IFN-α and β. Translation initiation depends on multiple factors including cap structure, poly-A tail, RNA secondary structures in 5′UTR, and AUG sequence context or Kozak sequence (Nelissen et al., 2012). When mRNA reaches the cytoplasm, a set of factors such as the trimeric eIF4F cap-binding complex binds the cap, wherein eIF4E interacts with the eIF4G and RNA helicase eIF4A. The poly A binding protein (PABP) on the 3′ poly-A tail interacts with eIF4G, circularizing the mRNA. After the formation of a 43S complex in 5′UTR near the cap, eIF4A helicase migrates with 43S complex in UTR to reach the start codon (Leppek et al., 2018; Müller-McNicoll and Neugebauer, 2013). Previous studies have shown that any stable secondary structures in the 5′UTRs can reduce translation initiation (Brierley et al., 2008; Beaudoin and Perreault, 2010; Kozak, 1994). In addition, another study has shown that 5′UTRs can improve translation regardless of 3′ UTR modification (Brierley et al., 2008). Thus, 5′UTR is one of the important factors that influence translation efficiency. Another approach for enhancing the stability and translation efficiency is to insert 3′UTRs derived alpha and beta-globin genes in IVT mRNA (Wang et al., 2013). Pyrimidine-rich element (PRE) in human 3′UTR beta-globin is a specific recognition site for RNA binding protein that was regulated mRNA stability (Russell and Liebhaber, 1996). In the present study, by using 5′ and 3′UTR of human beta-globin as well as other mentioned modifications, we enhanced the stability of GFPd2 IVT mRNA to 48 h, while the half-life of GFPd2 is between 2 and 10 h(Li et al., 1998). One of the factors that have the strongest effects on translation efficiency is AUG sequence context or Kozak sequence. The Kozak sequence is recognized by the ribosome as the translational start site. The amount of protein expression from a mRNA molecule is dependent on the strength of the Kozak sequence(Kozak, 1984). In the current study, for further improvement of mRNA translation, the Kozak sequence of the human beta-globin gene was replaced with the strongest Kozak sequence. Using 5′UTR of human beta-globin (with K1 sequence) and
5.1. Construction of in vitro transcription templates PUC57 vector containing 5′ and 3′UTR of human beta-globin, T7 promoter for in vitro transcription and the Kozak sequence was synthesized (by Biomatik corporation, Canada). The plasmid containing the destabilized GFP (pCAG-GFPd2) was a gift from Connie Cepko (Addgene plasmid # 14760). Primers for amplifying different DNA fragments, Splicing Overlap Extension PCR (SOEing PCR) and generation K2 sequence were designed using GeneRunner (Version 6.5.51 × 64 Beta). The specificity of the primers was confirmed by Primer-BLAST [www.ncbi.nlm.nih.gov/tools/primer-blast] (Table 1). The UTRs and GFPd2 fragments were separately amplified by the simple PCR method using Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs, U.K). The PCR products were determined on agarose gel electrophoresis and then recovered from the gel by AccuPrep® Gel Purification Kit (Bioneer, South Korea) according to the manufacturer's protocol. By the SOEing PCR method, 5′UTR was initially ligated to GFPd2 and then 3′UTR was ligated to 5′UTR-GFPd2 (5′UTR-GFPd2–3′UTR). The K2 sequence was constructed using the primers are given in Table 1 and the site-directed mutagenesis SOEing PCR method. The final recombinant constructs containing the K1 and K2 sequence were double digested using EcoRI (New England Biolabs, U·K) and BamHI (New England Biolabs, U·K) restriction enzymes and were then inserted into pcDNA™3.1 (+) Mammalian Expression Vector (Invitrogen) using Calcium chloride method. The recombinant plasmids were transformed into the Ecoli strain DH5α. The Colony PCR was performed to screen the plasmids containing the desired inserts. For further confirmation, DNA sequencing was conducted on both plasmids containing chimeric GFPd2. DNA template for in vitro transcription was prepared using Linearized plasmids in the 3′ end of the constructs and PCR amplification. The PCR reaction was performed in a thermal cycler with the following cycling conditions: 98 °C for 2 min, 32 cycles at 98 °C for 10s, 71 °C for 30 min, 72 °C for 1 min, and a final elongation step 234
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Fig. 3. Transfection efficiency using Lipofectamine 3000. a) pCAG-GFPd2 transfection in HFF cells as control of DNA transfection after 24 h; b) Plasmid DNA2 transfection in the HFF cells after 24 h; c) IVT mRNA2 transfection in the HFF cells after 24 h; d) IVT mRNA2 transfection in the HFF cells using trypsin protocol after 24 h; e) pCAG-GFPd2 transfection in HEK293T cells as control of DNA transfection after 24 h; f) Plasmid DNA1 transfection in HEK293T cells after 24 h; g) Plasmid DNA2 transfection in HEK293T cells after 24 h; h) IVT mRNA1 transfection in HEK293T cells after 24 h; i) IVT mRNA2 transfection in HEK293T cells after 3, 24, 36, 48, and 72 h. 235
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Fig. 4. The efficiency of GFP expression in transfected cells. a) The expression efficiency of plasmid DNAs and IVT mRNAs 24 h after transfection in HEK293T cells. Ten thousand gated events were collected per sample. b) The persistence of green fluorescent proteins using flow cytometry. Data represent means ± SEM. The percentage of positive HEK293T cells containing IVT mRNA2 remained stable between 24 and 72 h; in contrast, IVT mRNA1 decreased 24 h after transfection. c) Mean fluorescent intensity of GFP positive cells represents the protein level after IVT mRNA transfection. Overall mean fluorescent intensity of IVT mRNA2 was higher than IVT mRNA1. Plasmid K1: plasmid DNA with native Kozak sequence; Plasmid K2: plasmid DNA with modified Kozak sequence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
was quantified using a NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific,). The quality and size of mRNA were considered by 1% agarose gel electrophoresis. For the electrophoresis, the samples and PseudoU-RNA Molecular Weight Markers (Cellsript, USA) were loaded with 2× RNA loading dye (New England Biolabs, U·K) after a heat denaturation for 10 min at 65 °C followed by incubation on ice for 5 min. Subsequently, a cap1 structure was enzymatically added to the mRNA using ScriptCap™ m7G Capping System and ScriptCap™ 2′-OMethyltransferase Kit (Cellscript, USA) followed by addition of a Poly-A tail using A-Plus™ Poly(A) Polymerase Tailing Kit (Cellscript, USA) according to the manufacturer's protocols. RNA products were purified using RNA purification kit (Qiagen, Germany) and quantity and quality of mRNA were assessed as described above.
Fig. 5. Mean fluorescence intensity of GFP expression 24 h after transfection. The MFI of both plasmid DNAs was higher than IVT mRNAs and the MFI of IVT mRNA2 was higher than plasmid K1 and IVT mRNA1. Data represent means ± SEM.
5.3. Cell culture The HFF cells were cultured in DMEM F12 supplemented with 10% FBS (Gibco, USA) and incubated at 37 °C in 5% CO2. The HEK293T cells were cultured in DMEM high supplemented with 10% FBS (Gibco, USA) and incubated at 37 °C in 5% CO2. For transfection, the cells were in the 3rd passage.
72 °C for 5 min. 5.2. mRNA in vitro transcription The linearized plasmids or PCR products (1 μg) was used as a template for in vitro transcription using INCOGNITO™ T7 5mC- & Ψ-RNA Transcription Kit (Cellscript, USA) according to the manufacturer's protocols. The reaction time for IVT reaction was 3 h at 39 °C and then DNase I treatment was performed. RNA purification was performed using RNeasy Mini Kit (Qiagen, Germany) and concentration of mRNA
5.4. mRNAs and plasmid DNAs transfection The transfection was performed with Lipofectamine 3000 (Invitrogen). For transfection, the cells were seeded (5 × 104 HEK293T cells/well and 4 × 104 fibroblast cells/well) in 24-well plates a day 236
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Fig. 6. Cytotoxicity assay of the transfected cells with various IVT mRNAs and plasmid DNAs using transfection reagents. Cell viability of HEK293T cells 24 h after transfection was evaluated using MTT assay. Lipofectamine: the complex of Lipofectamine 3000 without construct; mRNA1 and plasmid1: IVT mRNA and plasmid with K1 sequence; mRNA2 and plasmid2: IVT mRNA and plasmid with K2 sequence.
Lipofectamine 3000-mRNA/DNA complex were divided into 24 well plates. 5.5. Toxicity assay For MTT assay, HEK293T cells were seeded in 96-well plates (1 × 104 cells/well) a day prior to the experiment and then Lipofectamine transfection was performed according to the manufacturer's protocol. The viability was evaluated 24 h post-transfection. The medium of the cells was removed and MTT made up in the medium to a final concentration of 0.5 mg/mL was added. After 3 h of incubation at 37 °C, the plate was centrifuged for 5 min in 3000 rpm and then MTT solution was aspirated and 100 μl/well DMSO was added. The plate was incubated at 37 °C for 30 min and the absorbance (570/630 nm) was measured using a microplate reader (BioTek, USA).
Fig. 7. Immune response activation following mRNA transfection.
prior to the experiment. The culture media was changed to Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) 2 h before all transfections. Transfection was performed according to the manufacturer's protocol and then the mixture was added to the culture media and incubated at 37 °C in 5% CO2. Six hours after transfection, the culture media was changed to either fibroblast or HEK293T cell medium. After transfection, the cells were monitored under a fluorescence microscope every 1 h, as long as was begun the expression of the green fluorescent protein, and was followed by every 4 h. The transfection of the HFF cells were performed on freshly trypsinized cells in suspension as well. The transfection complex was made according to the manufacturer's protocol. Subsequently, the mixture was added to the cell suspensions. After 20 min of incubation at room temperature, the cell and
5.6. Measurement of interferons by ELISA To determine the innate immune response following IVT mRNA transfection using Lipofectamine 3000, the level of hIFN-α and -β in the supernatant of transfected and non-transfected cells were measured by Human IFN- α and -β ELISA kit (ZellBio, Germany) according to the manufacturer's instructions. 5.7. Flow cytometry assay The transfection efficiency and GFP expression intensity of plasmid DNAs and IVT mRNAs in the HFF and HEK293T cells were evaluated by flow cytometry assay. In order to, the HEK293T cells were trypsinized
Table 1 Primer sequence for amplification of the fragments, SOEing PCR, and generation of K2 sequence. DNA fragment name 5′UTR
3′UTR GFPd2K1 GFPd2K2
Primer name 5′UF1 5′UR1 5′UF2 5′UR2 3′UF1 3′UR1 GK1F GK1R GK2F GK2R
Primer sequence 5′tcaaggatccGATCAATAATACGACTCACTATAG3′ 5′ctcaccATGGTGTCTGTTTGAGGTTG 3′ 5′tcaaggatccGATCAATAATACGACTCACTATAG 3′ 5′cttgctcaccatGGTGGCGGCTTGAGGTTG 3′ 5′caatgtgtagGCTCGCTTTCTTGCTGTCC 3′ 5′ cacagaattcGCTCTTCTTTTTGCAATG3′ 5′aacagacaccATGGTGAGCAAGGGCGAG 3′ 5′ gaaagcgagcCTACACATTGATCCTAGCAGAAG3′ 5′ gccaccATGGTGAGCAAGGGCGAG 3′ 5′ gaaagcgagcCTACACATTGATCCTAGCAGAAG 3′
Key: Overlap of the complementary sequence is shown in bold lowercase letters. 237
Product length (bp) 185 185 156 853 853
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24, 36, 48, and 72 h after transfection; however, the HFF cells were followed for the first 24 h post-transfection. The cells were washed twice with PBS and then suspended in PBS and analyzed by flow cytometry method (BD FACS Calibur).
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Stem Cell Res. 16 (3), 662–672. Ross, J., Sullivan, T.D., 1985. Half-lives of beta and gamma globin messenger RNAs and of protein synthetic capacity in cultured human reticulocytes. Blood. 66 (5), 1149–1154. Russell, J., Liebhaber, S.A., 1996. The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3'untranslated region. Blood. 87 (12), 5314–5323. Sonenberg, N., Hinnebusch, A.G., 2009. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell. 136 (4), 731–745. Van Der Velden, A.W., Thomas, A.A., 1999. The role of the 5′ untranslated region of an mRNA in translation regulation during development. Int. J. Biochem. Cell Biol. 31 (1), 87–106. Volloch, V., Housman, D., 1981. Stability of globin mRNA in terminally differentiating murine erythroleukemia cells. Cell. 23 (2), 509–514. Wang, Y., Su, H.-h., Yang, Y., Hu, Y., Zhang, L., Blancafort, P., et al., 2013. Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. Mol. Ther. 21 (2), 358–367. Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.-H., Li, H., Lau, F., et al., 2010. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7 (5), 618–630. Wickramasinghe, V.O., Laskey, R.A., 2015. Control of mammalian gene expression by selective mRNA export. Nat. Rev. Mol. Cell Biol. 16 (7), 431. Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A., et al., 1990. Direct gene transfer into mouse muscle in vivo. Science. 247 (4949), 1465–1469. Wu, B., Peisley, A., Richards, C., Yao, H., Zeng, X., Lin, C., et al., 2013. Structural basis for dsRNA recognition, filament formation, and antiviral signal activation by MDA5. Cell. 152 (1–2), 276–289. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., et al., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5 (7), 730. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., et al., 2005. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J. Immunol. 175 (5), 2851–2858. Zou, S., Scarfo, K., Nantz, M., Hecker, J., 2010. Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells. Int. J. Pharm. 389 (1–2), 232–243.
5.8. Statistical analysis All experiments were repeated at least three times. One-way analysis of variance (ANOVA) was used to determine whether there is any statistically significant difference between the experiments. The p values < 0.05 were considered statistically significant. Data were presented as mean ± SEM. Abbreviations IVT in vitro transcription GFPd2 destabilized green fluorescent protein or destabilized GFP SOEing PCR Splicing Overlap Extension PCR RNPs Ribonucleo proteins K1 native Kozak sequence of human beta-globin K2 modified Kozak sequence Acknowledgements The study team would like to gratefully acknowledge to the staff of Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran and Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran for their sincere cooperation. Funding The present article was extracted from a PhD thesis and supported by a National Institute for Medical Research Development (NIMAD) under a grant (number 958923), the Iranian National Science Foundation (INSF) under a grant (number 94808805), and Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Declaration of Competing Interest The authors declare no competing financial interests. References Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001a. Recognition of doublestranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature. 413 (6857), 732. Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001b. Recognition of doublestranded RNA and activation of NF-κB by toll-like receptor 3. Nature. 413 (6857), 732. Anderson, B.R., Muramatsu, H., Nallagatla, S.R., Bevilacqua, P.C., Sansing, L.H., Weissman, D., et al., 2010. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38 (17), 5884–5892. Asrani, K.H., Farelli, J.D., Stahley, M.R., Miller, R.L., Cheng, C.J., Subramanian, R.R., et al., 2018. Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA. RNA Biol. 0, 1–7. Babendure, J.R., Babendure, J.L., Ding, J.-H., Tsien, R.Y., 2006. Control of mammalian translation by mRNA structure near caps. Rna. 12 (5), 851–861. Balachandran, S., Roberts, P.C., Brown, L.E., Truong, H., Pattnaik, A.K., Archer, D.R., et al., 2000. Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity. 13 (1), 129–141. Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D., 2001. Spatial and temporal control of RNA stability. Proc. Natl. Acad. Sci. 98 (13), 7025–7028.
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Improved translation efficiency of therapeutic mRNA a
Farzaneh Zarghampoor , Negar Azarpira Ali Mohammad Foroughmanda a b c
b,⁎
a
T c
, Saeed Reza Khatami , Abbas Behzad-Behbahani ,
Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: UTR mRNA In vitro transcription Kozak sequence Beta-globin mRNA translation Green fluorescent protein
Recent developments in the field of the messenger RNA and its advantages versus DNA have led to a renewed interest in mRNA-based technologies. Despite its advantages, mRNA therapy has a number of drawbacks including low amount of mRNA production, short-term existence of mRNA and mRNA-mediated protein within the cell, severe mRNA cytotoxicity, and immune response activation following mRNA transfection. Here, we applied untranslated regions of human beta-globin to increase the stability and translation efficiency of a destabilized GFP mRNA. In order to suppress the innate immune response, which is the main barrier of mRNA therapy, we used the vaccinia virus derived capping enzyme and substituted standard nucleotides with modified nucleotides. At the end, the Kozak sequence of human beta-globin was replaced with the strongest sequence for the further improvement of mRNA translation. Overall, these modifications with native Kozak (K1) sequence of human beta-globin enhanced the stability of destabilized GFP mRNA up to 48 h and no increase in the level of interferon-α and -β was found. The GFP expression of mRNA with modified Kozak (K2) sequence initiated earlier than mRNA and plasmid DNA with K1 sequence. In contrast to mRNA with K1 sequence, the cells containing mRNA with K2 sequence remained positive for GFP expression up to 72 h post-transfection. Interestingly, transfection efficiency and mean fluorescence intensity (MFI) of mRNA with K2 sequence were higher than mRNA and plasmid DNA with K1 sequence. Taken together, these results provide valuable information for the optimization of mRNA stability and translation. Therefore, the methods used in the current study can successfully be applied for reprogramming, gene editing, trans-differentiation, tumour therapy, and gene therapy.
1. Background Nucleic acid-based therapy was first reported by Wolff et al. (1990). At that time, gene therapy was based on viral vectors and plasmid DNA technology. Recent advances in new treatment based on mRNA and its advantages versus DNA-based therapy make mRNA an attractive platform for gene therapy. Unlike DNA-based therapies, mRNA does not need to enter the nucleus; thus, for both non-dividing and slowly-dividing cells, mRNA therapy is more effective than DNA therapy (Zou et al., 2010). In addition, mRNA does not integrate into the host genome and there is no risk of insertional mutagenesis (Bernal, 2013; Hacein-Bey-Abina et al., 2003). Transient expression is another potential usefulness of mRNA therapy, which is important for the stability of the cells. However, mRNA-based therapy has several major problems: the short half-life of mRNA and mRNA-mediated protein, low level of in vitro transcription (IVT), severe mRNA cytotoxicity, and immune
⁎
response activation following mRNA transfection (Lu and Li, 2013). Mature mRNA in the eukaryotic cells contained several major regions that influence the degradation by exonucleases and translation efficiency (Leppek et al., 2018). Of those, 5′ and 3′ untranslated regions (UTRs) are known to play crucial roles in the regulation of translation (Van Der Velden and Thomas, 1999) and stability (Bashirullah et al., 2001). Therefore, one strategy that enhances translation efficiency and mRNA stability is using the variants of UTRs. To increase the stability of mRNA, 3′UTR of various endogenous mRNAs which have large amounts of protein production and long half-life such as α-globin, albumin and, β-globin has been studied extensively (Volloch and Housman, 1981; Ross and Sullivan, 1985). Thus, the use of 3′UTR represents a logical method to increase mRNA stability. Translation efficiency could potentially be influenced by the presence of the cap structure, 5′UTR sequence, and the Kozak sequence. In the eukaryotic cells, typically pre-initiation complex of translation is
Corresponding author. E-mail address: [email protected] (N. Azarpira).
https://doi.org/10.1016/j.gene.2019.05.008 Received 1 February 2019; Received in revised form 2 May 2019; Accepted 3 May 2019 Available online 04 May 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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transfection efficiency in HEK293T cells was higher than HFF cells 24 h post-transfection (Fig. 3; i 24 h and c). To improve IVT mRNA transfection of HFF cells, we trypsinized the cells and then transfection was done in suspension. The results showed that the efficiency of modified transfection was improved compared with the conventional method (Fig. 3; c and d). The GFP expression of IVT mRNA2 (with K2 sequence) in HEK293T cells was initiated 2-h post-transfections. The cells with the IVT mRNA1 (with K1 sequence) initiated GFP expression 6-h after transfection. In contrast to IVT mRNA2, the intensity of GFP expression of IVT mRNA1 was very low (Fig. 3; i 24 h and h). Overall, the transfection rate of IVT mRNAs was higher than plasmid DNA 24 h after incubation (Fig. 3; h vs f and i 24 h vs g). The intensity of GFP expression from IVT mRNA2 was decreased approximately 36 h after transfection (Fig. 3; i 36 h). However, the HEK293T cells were positive for GFP expression up to 72 h. In comparison with IVT mRNA2, the intensity of GFP expression from IVT mRNA1 was decreased approximately 24 h after transfection. The same results were obtained when the HFF cells were transfected with the constructs.
formed near of a cap structure and then scanning of the 5′UTR reaches the start codon. Thus, the 5′UTR plays an important role in the translation efficiency as it represents protein binding sites for a pre-initiation complex of translation and secondary structures that impact on scanning the 5′UTR (Sonenberg and Hinnebusch, 2009). Recent evidence suggests that the 5′UTR is the primary driver for protein translation from IVT mRNA and modification of the 5′UTR appears to have the largest impact on translation (Asrani et al., 2018). In the vertebrates, the most efficient Kozak sequence is gccRccAUGG (R = purine). The purine R is located in position −3 and G in position +4 have the strongest effect (Kozak, 1986; Babendure et al., 2006). Perhaps, the most serious disadvantage of mRNA-based technologies is the innate immune response. Exogenous RNAs with the signature of viral nucleic acids such as uncapped 5′ triphosphate, complex secondary structures in RNA such as pseudoknots, and GU-rich RNA are pathogen-associated molecular patterns for Toll-like receptors (Wu et al., 2013; Forsbach et al., 2008; Nallagatla et al., 2007). Therefore, IVT mRNAs with this signature may interact with pattern recognition receptors (PRRs), such as TLRs (Alexopoulou et al., 2001a; Karikó et al., 2004), RIG I, MDA5, PKR, OAS-L and RLR, and induce an innate immune response (Yoneyama et al., 2004; Yoneyama et al., 2005; Balachandran et al., 2000; Donovan et al., 2013). Stimulating the innate immune response can cause a variety of effects including cell cycle arrest, translation block, upregulation of PRRs, and apoptosis (Alexopoulou et al., 2001b). To improve the stability of IVT mRNA and reduce the innate immune response, we used 5′ and 3′UTR of human beta-globin, Vaccinia virus derived capping enzyme for generation cap 1 structure, substituted cytidine and uridine with 5-methylcytidine and pseudouridine, and performed polyadenylation in the 3′ end of IVT mRNA. In addition, for enhancing protein production of IVT mRNA, we replaced the Kozak sequence of human beta-globin with the strongest Kozak sequence. Finally, the translation efficiency of both constructs with K1 and K2 sequence was evaluated in human foreskin fibroblast (HFF) cell and HEK293T cell line.
2.4. Flow cytometry assay The transfection efficiency and GFP expression intensity of plasmid DNAs and IVT mRNAs in the HFF and HEK293T cells were evaluated by flow cytometry assay 24 h post-incubation. Overall, the transfection efficiency of IVT mRNAs was higher than plasmid DNAs; however, the mean fluorescence intensity of each plasmid DNA was higher than the related IVT mRNA. In comparison with IVT mRNA1, transfection efficiency and the mean fluorescence intensity of IVT mRNA2 were higher. In addition, the stability and GFP expression intensity of both IVT mRNAs were monitored in the HEK293T cells 24, 36, 48, and 72 h after transfection. Maximal GFP expression was determined between 24 and 48 h after transfection. The percentage of GFP positive cells containing IVT mRNA2 remained stable 24 to 72 h and decreased 72 h after transfection. In contrast, the percentage of GFP positive cells containing IVT mRNA1 decreased 24 h post-transfection (Figs. 4 and 5).
2. Results 2.5. Cytotoxicity assay of plasmid DNAs, IVT mRNAs, and transfection reagents
2.1. Construction of recombinant plasmids As illustrated in Fig. 1, 5′UTR of human beta-globin (185 bp) with K1 sequence (ACAGACACCATG), 3′UTR of human beta-globin (156 bp) and destabilized GFP (GFPd2, 853 bp) were amplified by simple PCR. Following this, the DNA fragments were ligated together by SOEing PCR [5′UTR-K1-GFPd2–3′UTR]. Finally, the construct with K2 sequence (GCCGCCACCATG) was generated using the new primers for 3′ end of 5′UTR and 5′ end of GFPd2 (Fig. 1).
The effect of Lipofectamine 3000 reagent, plasmid DNAs, and IVT mRNAs on the cell viability were evaluated using MTT assay. As shown in Fig. 6, plasmid DNA tends to cause greater cell toxicity when paired with Lipofectamine 3000. However, there were no statistically significant differences between the viability of transfected and nontransfected cells after 24 h (Fig. 6). 2.6. Immune response stimulation following IVT mRNA transfection
2.2. mRNA in vitro transcription To determine the innate immune response following IVT mRNA transfection using Lipofectamine 3000, we measured the level of human interferon alpha and beta (hIFN-α/β) in the supernatant of HEK293T cells by ELISA. The concentration of hIFN-β (75.181 pg/ml) was detected some higher than hIFN-α (58.239 pg/ml). However, in comparison with non-transfected cells, no significant differences were observed in the levels of hIFN-α and -β in the supernatant of the transfected cells (Fig. 7).
For in vitro transcription, linearized plasmid DNA and the PCR product were used. Interestingly, the efficiency of in vitro transcription from a PCR product (65 μg RNA from 1 μg DNA) was higher than linearized plasmid DNA (8 μg RNA from 1 μg DNA). Furthermore, IVT mRNA produced from linearized plasmid DNA did not have any GFP expression in the HEK293T cell line. Hence, for in vitro transcription, we used PCR product as template. The quality and size of mRNAs were assessed on agarose gel electrophoresis (Fig. 2). In addition, the presence of a poly-A tail in the 3′end of IVT mRNA was confirmed with agarose gel electrophoresis.
3. Discussion IVT mRNA is a multifunctional molecule that can be applied for reprogramming, gene editing, transdifferentiation, tumour therapy, vaccination, and gene therapy. In recent years, researchers have shown an increased interest in IVT mRNA due to important differences between IVT mRNA and plasmid DNA. Compared with plasmid DNA, IVT mRNA has a transient nature; therefore, it probably offers improved
2.3. IVT mRNA and plasmid DNA transfection To assess the protein expression efficiency of K1 and K2 sequences, we transfected IVT mRNA and the plasmid DNA in the HFF and HEK293T cells. As shown in Fig. 3, using fluorescent microscopy, the 232
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Fig. 1. Construction of recombinant plasmids. (a) 5′UTR of human beta-globin containing K1 and K2 sequence was amplified using simple PCR. The 3′UTR of betaglobin and GFPd2 were amplified by simple PCR, as well. (b) The DNA fragments were spliced together, using SOEing PCR. (c) Subsequently, both of the constructs with K1 and K2 sequences were inserted into pcDNA3.1+ vector by calcium chloride method. (d) Agarose gel electrophoresis of simple and SOEing PCRs; (1) 100 bp DNA ladder, (2 and 3) 5′UTR of human beta-globin with K1 and K2 sequence (185 bp), (4) 3′UTR of human beta-globin (156 bp), (5) GFPd2 fragment (853 bp), (6) the SOEing PCR1 product (5′UTR-GFPd2, 1011 bp), and (7) the SOEing PCR2 product (5′UTR-GFPd2–3′UTR, 1147 bp). K1: native Kozak sequence; K2: modified Kozak sequence; Construct1: 5′UTR-K1-GFPd2–3′UTR; Construct2: 5′UTR- K2-GFPd2–3′UTR.
complex. Additionally, IVT mRNA is often delivered into the cell through the endolysosomal process. Therefore, the presence of abnormal IVT mRNA in the extracellular, endosomal, and cytoplasmic compartment, particularly the 5′ triphosphate mRNA, led to its detection by PRRs (such as PKR and RIGI). The PKR pathway is the most harmful antiviral pathway that leads to inhibition of protein expression from endogenous and exogenous mRNA. Activation of PRRs leads to the initiation of a cascade signalling that results in up-regulation of type I interferon and NF-kB related genes. On the other hand, Translation in the eukaryotic cells almost begins in the cap-dependent mechanism. Consequently, a key step in processing IVT mRNA is the addition of a cap structure into the 5′ end of mRNA. There are different capping systems to incorporate the cap structure in the 5′ end of IVT mRNA. To suppress the innate immune response, Warren et al. used cap analog ARCA and substituted modified nucleotides with the unmodified counterpart (substitution of cytidine and uridine with 5-methylcytidine and pseudouridine) (Warren et al., 2010). However, still the residual up-regulation of some targets of the interferon on the gene expression level was detected. Other studies have shown that the substitution of uridine with pseudouridine, suppresses the innate immune response and increases the protein expression (Anderson et al., 2010). In the present study, to suppress the immune response and increase translation, we used vaccinia virus derived capping enzyme to incorporate a cap1 structure into the 5′ end of IVT mRNA. The efficiency of this capping system is nearly 100% whereas efficiency cap analog ARCA is ~80% and cap analog is ~50% of IVT, and need RNA products purified or treated with phosphatase to eliminate any remaining 5′triphosphates. Additionally, we substituted cytidine and uridine with 5-methylcytidine and pseudouridine,
control over protein expression kinetics and nucleic acid dosing. For some cell types, such as non-dividing or slowly-dividing cells, IVT mRNA leads to more effective protein production (Zou et al., 2010; Wang et al., 2013). Furthermore, IVT mRNA remains in the cytoplasm and does not enter the nucleus; hence, there is no risk of integration into the host genome (Bernal, 2013; Hacein-Bey-Abina et al., 2003). Despite its safety and efficacy, IVT mRNA has several major drawbacks which should be overcome. One of the problems of IVT mRNA is the deficiency for scalable production of in vitro transcribed RNA. As previous studies have shown, the chemical in vitro RNA synthesis is inappropriate for large production of long templates (> 20nts) (Lu and Li, 2013; Nelissen et al., 2012). Therefore, in the present study for the large production of IVT mRNA using T7 RNA polymerase, linearized plasmid or PCR amplicon was used as the template. In comparison with the linearized plasmid, higher yield of mRNA was synthesized when the PCR amplified DNA was used as a template for in vitro transcription. The same scale of in vitro transcription has been reported when the PCR amplicon was used as a template for the large scale production of mRNA (Rohani et al., 2016). Although the reason has not been clearly described, the quality and purity of linearized plasmid may be the main reason for the low efficiency of in vitro transcription. Degradation of linearized plasmid or contamination with RNase during purification may have an adverse effect on the yield of in vitro transcription. Due to the large size of plasmid DNA molecules, it could be possible to damage the linearized plasmid DNA during purification in comparison with PCR amplicon. The main limitations of IVT mRNA for therapeutic applications are low stability and innate immune stimulation. Unlike IVT mRNA, endogenous mRNA is almost entirely in RNPs (Ribonucleo proteins) 233
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other modifications increased the stability and translation of GFPd2 to 48 h after transfection. However, the percentage of positive cells containing IVT mRNA with K2 sequence remained stable up to 72 h after transfection. On the other hands, the transfected cells with IVT mRNA2 initiated GFP expression earlier than IVT mRNA1. It is interesting to note that the amount of protein expression from each plasmid DNA was higher than IVT mRNA respective. However, the amount of protein expression from IVT mRNA2 was higher than plasmid DNA and IVT mRNA1. These results were not due to the higher stability of IVT mRNA2 than IVT mRNA1, in fact, the performance of modified Kozk sequence and enhancing in protein translation efficiency are the two main reasons. As previously mentioned, the translation of mRNA is a complex process involving multiple protein-protein and protein-mRNA interactions. These RNA binding proteins bind to the endogenous mRNA prior to export from the nucleus and can impact protein production (Wickramasinghe and Laskey, 2015). While IVT mRNA directly is delivered to the cytosol and would bypass these traditional modifications. This may explain the differences in time of initiation of GFP expression and relative protein expression from plasmid DNA and IVT mRNA. 4. Conclusions In summary, the current study provides important information to increase the stability and efficiency of mRNA translation, to suppress the immune response that main of hurdles of IVT mRNA, and highlight the main role of 5′UTR in enhancing protein production. In addition, using the modified Kozak sequence in the human 5′UTR beta-globin had a positive impact on the translation efficiency of IVT mRNA which is the new finding of this study.
Fig. 2. Agarose gel electrophoresis of heat-denatured IVT mRNA products. (1) Pseudouridine RNA Molecular Weight Markers, (2) IVT mRNA without cap and poly-A tail, (3): IVT mRNA with cap1 and poly-A tail.
5. Materials and methods
respectively. Hence, in supernatant transfected cells no increases were detected in IFN-α and β. Translation initiation depends on multiple factors including cap structure, poly-A tail, RNA secondary structures in 5′UTR, and AUG sequence context or Kozak sequence (Nelissen et al., 2012). When mRNA reaches the cytoplasm, a set of factors such as the trimeric eIF4F cap-binding complex binds the cap, wherein eIF4E interacts with the eIF4G and RNA helicase eIF4A. The poly A binding protein (PABP) on the 3′ poly-A tail interacts with eIF4G, circularizing the mRNA. After the formation of a 43S complex in 5′UTR near the cap, eIF4A helicase migrates with 43S complex in UTR to reach the start codon (Leppek et al., 2018; Müller-McNicoll and Neugebauer, 2013). Previous studies have shown that any stable secondary structures in the 5′UTRs can reduce translation initiation (Brierley et al., 2008; Beaudoin and Perreault, 2010; Kozak, 1994). In addition, another study has shown that 5′UTRs can improve translation regardless of 3′ UTR modification (Brierley et al., 2008). Thus, 5′UTR is one of the important factors that influence translation efficiency. Another approach for enhancing the stability and translation efficiency is to insert 3′UTRs derived alpha and beta-globin genes in IVT mRNA (Wang et al., 2013). Pyrimidine-rich element (PRE) in human 3′UTR beta-globin is a specific recognition site for RNA binding protein that was regulated mRNA stability (Russell and Liebhaber, 1996). In the present study, by using 5′ and 3′UTR of human beta-globin as well as other mentioned modifications, we enhanced the stability of GFPd2 IVT mRNA to 48 h, while the half-life of GFPd2 is between 2 and 10 h(Li et al., 1998). One of the factors that have the strongest effects on translation efficiency is AUG sequence context or Kozak sequence. The Kozak sequence is recognized by the ribosome as the translational start site. The amount of protein expression from a mRNA molecule is dependent on the strength of the Kozak sequence(Kozak, 1984). In the current study, for further improvement of mRNA translation, the Kozak sequence of the human beta-globin gene was replaced with the strongest Kozak sequence. Using 5′UTR of human beta-globin (with K1 sequence) and
5.1. Construction of in vitro transcription templates PUC57 vector containing 5′ and 3′UTR of human beta-globin, T7 promoter for in vitro transcription and the Kozak sequence was synthesized (by Biomatik corporation, Canada). The plasmid containing the destabilized GFP (pCAG-GFPd2) was a gift from Connie Cepko (Addgene plasmid # 14760). Primers for amplifying different DNA fragments, Splicing Overlap Extension PCR (SOEing PCR) and generation K2 sequence were designed using GeneRunner (Version 6.5.51 × 64 Beta). The specificity of the primers was confirmed by Primer-BLAST [www.ncbi.nlm.nih.gov/tools/primer-blast] (Table 1). The UTRs and GFPd2 fragments were separately amplified by the simple PCR method using Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs, U.K). The PCR products were determined on agarose gel electrophoresis and then recovered from the gel by AccuPrep® Gel Purification Kit (Bioneer, South Korea) according to the manufacturer's protocol. By the SOEing PCR method, 5′UTR was initially ligated to GFPd2 and then 3′UTR was ligated to 5′UTR-GFPd2 (5′UTR-GFPd2–3′UTR). The K2 sequence was constructed using the primers are given in Table 1 and the site-directed mutagenesis SOEing PCR method. The final recombinant constructs containing the K1 and K2 sequence were double digested using EcoRI (New England Biolabs, U·K) and BamHI (New England Biolabs, U·K) restriction enzymes and were then inserted into pcDNA™3.1 (+) Mammalian Expression Vector (Invitrogen) using Calcium chloride method. The recombinant plasmids were transformed into the Ecoli strain DH5α. The Colony PCR was performed to screen the plasmids containing the desired inserts. For further confirmation, DNA sequencing was conducted on both plasmids containing chimeric GFPd2. DNA template for in vitro transcription was prepared using Linearized plasmids in the 3′ end of the constructs and PCR amplification. The PCR reaction was performed in a thermal cycler with the following cycling conditions: 98 °C for 2 min, 32 cycles at 98 °C for 10s, 71 °C for 30 min, 72 °C for 1 min, and a final elongation step 234
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Fig. 3. Transfection efficiency using Lipofectamine 3000. a) pCAG-GFPd2 transfection in HFF cells as control of DNA transfection after 24 h; b) Plasmid DNA2 transfection in the HFF cells after 24 h; c) IVT mRNA2 transfection in the HFF cells after 24 h; d) IVT mRNA2 transfection in the HFF cells using trypsin protocol after 24 h; e) pCAG-GFPd2 transfection in HEK293T cells as control of DNA transfection after 24 h; f) Plasmid DNA1 transfection in HEK293T cells after 24 h; g) Plasmid DNA2 transfection in HEK293T cells after 24 h; h) IVT mRNA1 transfection in HEK293T cells after 24 h; i) IVT mRNA2 transfection in HEK293T cells after 3, 24, 36, 48, and 72 h. 235
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Fig. 4. The efficiency of GFP expression in transfected cells. a) The expression efficiency of plasmid DNAs and IVT mRNAs 24 h after transfection in HEK293T cells. Ten thousand gated events were collected per sample. b) The persistence of green fluorescent proteins using flow cytometry. Data represent means ± SEM. The percentage of positive HEK293T cells containing IVT mRNA2 remained stable between 24 and 72 h; in contrast, IVT mRNA1 decreased 24 h after transfection. c) Mean fluorescent intensity of GFP positive cells represents the protein level after IVT mRNA transfection. Overall mean fluorescent intensity of IVT mRNA2 was higher than IVT mRNA1. Plasmid K1: plasmid DNA with native Kozak sequence; Plasmid K2: plasmid DNA with modified Kozak sequence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
was quantified using a NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific,). The quality and size of mRNA were considered by 1% agarose gel electrophoresis. For the electrophoresis, the samples and PseudoU-RNA Molecular Weight Markers (Cellsript, USA) were loaded with 2× RNA loading dye (New England Biolabs, U·K) after a heat denaturation for 10 min at 65 °C followed by incubation on ice for 5 min. Subsequently, a cap1 structure was enzymatically added to the mRNA using ScriptCap™ m7G Capping System and ScriptCap™ 2′-OMethyltransferase Kit (Cellscript, USA) followed by addition of a Poly-A tail using A-Plus™ Poly(A) Polymerase Tailing Kit (Cellscript, USA) according to the manufacturer's protocols. RNA products were purified using RNA purification kit (Qiagen, Germany) and quantity and quality of mRNA were assessed as described above.
Fig. 5. Mean fluorescence intensity of GFP expression 24 h after transfection. The MFI of both plasmid DNAs was higher than IVT mRNAs and the MFI of IVT mRNA2 was higher than plasmid K1 and IVT mRNA1. Data represent means ± SEM.
5.3. Cell culture The HFF cells were cultured in DMEM F12 supplemented with 10% FBS (Gibco, USA) and incubated at 37 °C in 5% CO2. The HEK293T cells were cultured in DMEM high supplemented with 10% FBS (Gibco, USA) and incubated at 37 °C in 5% CO2. For transfection, the cells were in the 3rd passage.
72 °C for 5 min. 5.2. mRNA in vitro transcription The linearized plasmids or PCR products (1 μg) was used as a template for in vitro transcription using INCOGNITO™ T7 5mC- & Ψ-RNA Transcription Kit (Cellscript, USA) according to the manufacturer's protocols. The reaction time for IVT reaction was 3 h at 39 °C and then DNase I treatment was performed. RNA purification was performed using RNeasy Mini Kit (Qiagen, Germany) and concentration of mRNA
5.4. mRNAs and plasmid DNAs transfection The transfection was performed with Lipofectamine 3000 (Invitrogen). For transfection, the cells were seeded (5 × 104 HEK293T cells/well and 4 × 104 fibroblast cells/well) in 24-well plates a day 236
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Fig. 6. Cytotoxicity assay of the transfected cells with various IVT mRNAs and plasmid DNAs using transfection reagents. Cell viability of HEK293T cells 24 h after transfection was evaluated using MTT assay. Lipofectamine: the complex of Lipofectamine 3000 without construct; mRNA1 and plasmid1: IVT mRNA and plasmid with K1 sequence; mRNA2 and plasmid2: IVT mRNA and plasmid with K2 sequence.
Lipofectamine 3000-mRNA/DNA complex were divided into 24 well plates. 5.5. Toxicity assay For MTT assay, HEK293T cells were seeded in 96-well plates (1 × 104 cells/well) a day prior to the experiment and then Lipofectamine transfection was performed according to the manufacturer's protocol. The viability was evaluated 24 h post-transfection. The medium of the cells was removed and MTT made up in the medium to a final concentration of 0.5 mg/mL was added. After 3 h of incubation at 37 °C, the plate was centrifuged for 5 min in 3000 rpm and then MTT solution was aspirated and 100 μl/well DMSO was added. The plate was incubated at 37 °C for 30 min and the absorbance (570/630 nm) was measured using a microplate reader (BioTek, USA).
Fig. 7. Immune response activation following mRNA transfection.
prior to the experiment. The culture media was changed to Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) 2 h before all transfections. Transfection was performed according to the manufacturer's protocol and then the mixture was added to the culture media and incubated at 37 °C in 5% CO2. Six hours after transfection, the culture media was changed to either fibroblast or HEK293T cell medium. After transfection, the cells were monitored under a fluorescence microscope every 1 h, as long as was begun the expression of the green fluorescent protein, and was followed by every 4 h. The transfection of the HFF cells were performed on freshly trypsinized cells in suspension as well. The transfection complex was made according to the manufacturer's protocol. Subsequently, the mixture was added to the cell suspensions. After 20 min of incubation at room temperature, the cell and
5.6. Measurement of interferons by ELISA To determine the innate immune response following IVT mRNA transfection using Lipofectamine 3000, the level of hIFN-α and -β in the supernatant of transfected and non-transfected cells were measured by Human IFN- α and -β ELISA kit (ZellBio, Germany) according to the manufacturer's instructions. 5.7. Flow cytometry assay The transfection efficiency and GFP expression intensity of plasmid DNAs and IVT mRNAs in the HFF and HEK293T cells were evaluated by flow cytometry assay. In order to, the HEK293T cells were trypsinized
Table 1 Primer sequence for amplification of the fragments, SOEing PCR, and generation of K2 sequence. DNA fragment name 5′UTR
3′UTR GFPd2K1 GFPd2K2
Primer name 5′UF1 5′UR1 5′UF2 5′UR2 3′UF1 3′UR1 GK1F GK1R GK2F GK2R
Primer sequence 5′tcaaggatccGATCAATAATACGACTCACTATAG3′ 5′ctcaccATGGTGTCTGTTTGAGGTTG 3′ 5′tcaaggatccGATCAATAATACGACTCACTATAG 3′ 5′cttgctcaccatGGTGGCGGCTTGAGGTTG 3′ 5′caatgtgtagGCTCGCTTTCTTGCTGTCC 3′ 5′ cacagaattcGCTCTTCTTTTTGCAATG3′ 5′aacagacaccATGGTGAGCAAGGGCGAG 3′ 5′ gaaagcgagcCTACACATTGATCCTAGCAGAAG3′ 5′ gccaccATGGTGAGCAAGGGCGAG 3′ 5′ gaaagcgagcCTACACATTGATCCTAGCAGAAG 3′
Key: Overlap of the complementary sequence is shown in bold lowercase letters. 237
Product length (bp) 185 185 156 853 853
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24, 36, 48, and 72 h after transfection; however, the HFF cells were followed for the first 24 h post-transfection. The cells were washed twice with PBS and then suspended in PBS and analyzed by flow cytometry method (BD FACS Calibur).
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5.8. Statistical analysis All experiments were repeated at least three times. One-way analysis of variance (ANOVA) was used to determine whether there is any statistically significant difference between the experiments. The p values < 0.05 were considered statistically significant. Data were presented as mean ± SEM. Abbreviations IVT in vitro transcription GFPd2 destabilized green fluorescent protein or destabilized GFP SOEing PCR Splicing Overlap Extension PCR RNPs Ribonucleo proteins K1 native Kozak sequence of human beta-globin K2 modified Kozak sequence Acknowledgements The study team would like to gratefully acknowledge to the staff of Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran and Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran for their sincere cooperation. Funding The present article was extracted from a PhD thesis and supported by a National Institute for Medical Research Development (NIMAD) under a grant (number 958923), the Iranian National Science Foundation (INSF) under a grant (number 94808805), and Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Declaration of Competing Interest The authors declare no competing financial interests. References Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001a. Recognition of doublestranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature. 413 (6857), 732. Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001b. Recognition of doublestranded RNA and activation of NF-κB by toll-like receptor 3. Nature. 413 (6857), 732. Anderson, B.R., Muramatsu, H., Nallagatla, S.R., Bevilacqua, P.C., Sansing, L.H., Weissman, D., et al., 2010. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38 (17), 5884–5892. Asrani, K.H., Farelli, J.D., Stahley, M.R., Miller, R.L., Cheng, C.J., Subramanian, R.R., et al., 2018. Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA. RNA Biol. 0, 1–7. Babendure, J.R., Babendure, J.L., Ding, J.-H., Tsien, R.Y., 2006. Control of mammalian translation by mRNA structure near caps. Rna. 12 (5), 851–861. Balachandran, S., Roberts, P.C., Brown, L.E., Truong, H., Pattnaik, A.K., Archer, D.R., et al., 2000. Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity. 13 (1), 129–141. Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D., 2001. Spatial and temporal control of RNA stability. Proc. Natl. Acad. Sci. 98 (13), 7025–7028.
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