The development of new treatments in the post-genomic era requires methods for safe delivery of foreign genetic information in vivo. As a transient, natural and controllable alternative to recombinant viruses or plasmid DNA (pDNA), purified or in vitro transcribed messenger RNA (mRNA) can be used for the expression of any therapeutic protein in vitro and in vivo. As it has been shown previously, the simple injection of naked mRNA results in local uptake and expression. We show here that this process, in the skin, can greatly be modulated according to the injection solution composition and blocked by an excess of competing nucleic acids or a drug affecting cytosolic mobility. Different cell types at the site of injection can take up the foreign nucleic acid molecules and the protein translated from this is detected for no more than a few days. To test this gene transfer method in humans, we produced in vitro transcribed mRNA under good manufacturing practice (GMP) conditions in a dedicated facility. After injection into the human dermis, we could document the translation of the exogenous mRNA. Our results pave the way toward the use of mRNA as a vehicle for transient gene delivery in humans.
Messenger RNA (mRNA) is a transient copy of the coding genomic information in any living organism. Its potential as a gene therapy vehicle was hindered, on the one hand, because of the technical difficulties and cost of mRNA production in research laboratories and, on the other because of the general belief that ubiquitous intracellular and extracellular RNases would quickly destroy the molecule and limit its efficacy as a genetic tool in vivo. However, Wolff et al.1 have shown 16 years ago that, in mice, the injection of naked genetic information in the form of plasmid DNA (pDNA) or mRNA can lead to protein expression. Following these results, many studies were undertaken which demonstrated that naked pDNA can be used for vaccination.2, 3 mRNA was rarely exploited until the late nineties when Gilboa's group showed that adoptive transfer of mRNA-transfected dendritic cells (DCs) primes a T-cell immune response.4 But, the direct injection of naked mRNA for vaccination or gene complementation remained quite unexplored, being reported in only four articles from three different teams5, 6, 7, 8 (for a review on mRNA-based vaccines, see Pascolo9). Because of the safety (minimal vector, completely and naturally catabolized in several hours), ease (no need for infrastructures for cell culture) and versatility (any mRNA of interest can be produced) of naked mRNA-based therapies compared to DNA-based therapies,10, 11 we decided to study and improve the intradermal delivery of this genetic vehicle. As shown in Figure 1, using intradermal injection of globin UTR-stabilized (RNActive, CureVac GmbH, Tübingen, Germany) luciferase-encoding mRNA in the ear pinna,12 we could investigate the effect of the injection solution composition on the efficacy of in vivo mRNA transfer (for method details, see Supplementary Information and Supplementary Figure S1). We tested the injection solutions used by authors of the few published works that were phosphate-buffered saline (PBS) and Hepes/NaCl, and in addition we tested Ringer lactate, which is a standard injection solution in humans. Figure 1a shows that RNActive dissolved in Ringer lactate gives a significant higher amount of luciferase expression than mRNA resuspended in Hepes/NaCl or PBS. Ringer lactate and PBS have three differences: only the former contains lactate and calcium, whereas the latter contains phosphate. We tested which of the two ions present in Ringer lactate is responsible for the observed enhanced mRNA transfer. Self-made Ringer without lactate or calcium was compared to self-made Ringer lactate. As shown in Figure 1b, although Ringer without lactate gave luciferase expression comparable to that observed with complete Ringer lactate, the absence of calcium in the injection solution lowered significantly (P=0.004) the efficiency of vector transfer to a level similar to PBS or Hepes/NaCl. When injecting naked mRNA solubilized in solutions with higher or lower concentrations of calcium compared to the standard Ringer lactate, we did not see higher amounts of luciferase protein (data not shown). Similarly, higher or lower Ringer lactate concentrations in the final injected solution did not increase the amount of luciferase proteins detected in the ear (Supplementary Information and Supplementary Figure S2). The quantity of luciferase produced by injection of RNActive in the skin can be higher than that obtained when transfecting primary cells using optimal electroporation as described13 (Figure 1c). Thus, the uptake and expression of injected naked RNActive is an efficacious process. Using Ringer lactate, we studied the kinetics of pDNA and mRNA expression after injection. As shown in Figure 1d, the amount of luciferase detected after RNActive injection peaks at ca. 17 h and is undetectable after 3 days. In comparison, pDNA injection gave a delayed protein expression that peaks at 3 days after injection and eventually lasted for more than 50 days. Of note, at the peak of expression, RNActive can give more protein expression than pDNA. The results were confirmed when we studied the luciferase expression using an optical imaging system (Figure 1e). This technology demonstrates that luciferase activity is detectable only at the site of RNActive injection. Thus, after intradermal injection, neither the RNActive nor the cells that take it up migrate to a distant site. Checking the effect of RNA concentration, we observed a linear increase in luciferase expression when increasing the RNActive amount from 1 to 5 μg in 100 μl injection volume (Figure 1f). Surprisingly, from 5 μg up to 80 μg of RNActive in 100 μl Ringer lactate, no change in the amount of luciferase activity was observed (Figure 1f). These results are different to those reported by Wolf et al. where the mRNA was injected intramuscularly. In Wolf's report, the more mRNA was injected, the more luciferase activity was detected. In contrast, our results indicate that the uptake of naked RNActive in the dermis is a saturable process. Therefore, these results suggest that at least one step in the uptake of exogenous RNActive by some cells of the dermis is mediated by an active biological mechanism. To investigate this mechanism, we studied the capacity of different macromolecules to compete with the RNActive uptake. A mass excess of irrelevant RNActive (coding Influenza matrix M1), fragmented (sonicated) luciferase-coding RNActive, dsRNA, pDNA, CpG DNA oligonucleotide or peptide was mixed with 10 μg of RNActive-coding luciferase and injected in the ear pinna. As shown in Figure 1g, the amount of resulting luciferase molecules was not changed when a peptide was added to the RNActive, but was significantly reduced (P<0.001 for CpG oligonucleotide, pDNA and irrelevant mRNA, P=0.017 for sonicated luc mRNA and P=0.04 for dsRNA) when an excess of any type of nucleic acid was added. Thus, the uptake-limiting step for injected RNActive's expression is a mechanism specific for nucleic acids. We then investigated further this active pathway that allows RNActive to reach the cytosol by using available inhibitors that block macropinocytose (Amiloride), clathrin-coated pits (Chlorpromazine), Caveolae (Nystatin) or depolymerization of actin and thus cytosolic mobility (Cytochalasin B). Each of these inhibitors is toxic and we used the dose reported to be efficacious and not lethal in vivo in mice.13, 14, 15, 16 Under these conditions, only Cytochalasin B significantly inhibited the expression of luciferase from injected RNActive (Figure 1h). Thus, it appears that the exogenous RNA goes through moving vesicles before being delivered into the cytosol. However, the lack of a statistically significant effect of Amiloride, Chlorpromazine and Nystatin may be due to too low dosage, poor availability of the drug at the site of RNA injection or unknown side effects. Thus, these experiments do not definitively rule out the involvement of macropinocytose, clathrin-coated pits and caveolae, respectively in the uptake of exogenous mRNA. We conclude from these experiments that the local uptake of exogenous RNA by cells in the dermis can be enhanced by the addition of calcium, is mediated by a saturable mechanism that is specific for nucleic acids and involves movements of vesicles. The expression of the transgene in the form of RNActive is transient: using this method, a sustained topical expression of a foreign molecule would require reinjection of RNActive approximately every 3 days. In contrast to pDNA or recombinant viruses that can show uncontrolled persistence, no accumulation or overexpression of the transgene-encoded protein could occur even with repeated mRNA applications.
In order to identify the cell type(s) that take up the exogenous RNA at the site of injection, we used β-galactosidase-coding RNActive. Approximately 16 h after injection, ears were frozen in embedding medium and cut into sections using a cryomicrotom. Sections were incubated with X-gal containing solution (for the optimization of the staining conditions, see Supplementary Figures S3–S8). Cells that had taken up exogenous RNA were identified by blue staining in sections (Figure 2a). Starting from the basal to the apical part of the ear, we visualized sections and documented the presence of blue cells. As shown in the diagram of Figure 2b, from one to ten blue cells could be observed in consecutive sections spanning more than 1 mm at the site of injection (starting at 3.00 mm from the base of the ear to 4.2 mm from the base of the ear). The partial section shown in panel a is the one located at 3720 μm from the top of the ear, it is labeled α. The characterization of blue cells by antibody staining was difficult because the blue color interferes strongly with both immunohistochemistry staining and fluorescence in different confocal microscopy channels (see Supplementary Figures S5–S7). Thus we tested other dyes metabolized by β-galactosidase and identified magenta red as a sensitive method that is compatible with fluorescence emission at 570 nm (Alexa red, see Supplementary Figure S6). Using an anti-mouse major histocompatibility complex (MHC) class II molecule antibody, we could demonstrate that most β-galactosidase-positive cells are not MHC class II positive. Two such stained sections (shown in parts) with the corresponding cells labeled β and χ are shown in Figure 2b and c. From the location of the cells, their shape and their MHC class II-negative phenotype, we conclude that cells that take up naked RNA at the site of injection are muscle cells, fibroblasts or keratinocytes (see also Supplementary Figures S7 and S8). For some cells expressing β-galactosidase, it could not be excluded that they are MHC class II positive as shown for cells β(2/8) and β(7/8) in Figure 2c, but most MHC class II-positive cells were negative for β-galactosidase expression. However, since antigen-presenting cells (APCs) such as Langerhans cells are very efficient in the degradation of proteins, our results do not exclude that APCs take up the foreign naked RNA and present peptides derived from the neotranslated antigen to the immune system.
To check that injected RNActive molecules are also taken up and expressed in human skin and in order to exploit this feature for therapy, we created the facilities, methods and quality management system that allow the production of high amounts of RNActive under good manufacturing practice (GMP) conditions. A purification step called PUREmessenger was implemented whereby the long RNA molecules are purified according to their length. This technology allows the elimination of contaminating shorter or longer transcripts. The final RNA is a highly pure mRNA transcript. We produced in this infrastructure pharmaceutical-grade RNActive coding for luciferase. The GMP quality RNA resuspended in Ringer lactate and injected in mice gave a significantly higher production of luciferase compared to the laboratory-grade mRNA (Figure 3a). A qualified healthy individual volunteered for intradermal injections of 150 μl of a Ringer lactate solution containing the GMP quality RNActive coding for luciferase. Before performing the experiment, a letter of consent was signed by the volunteer. Sixteen hours after injection, 3 mm diameter punch biopsies were performed under local anesthesia with 1% lidocainhydrochlorid (Xylocain). A biopsy made on the middle of the injection site (‘injected’ in Figure 3b) and one made outside the injection area (‘not injected’ in Figure 3b) were snap frozen, crushed and resuspended in a lysis buffer. As shown in Figure 3b, luciferase activity was found in the biopsy taken from the injection site. This result demonstrates the uptake of RNActive in vivo in the human skin. Since only a 3 mm punch biopsy – approximately 5 mm2 of skin – was performed in the injected area of approximately 300 mm2 (the diameter of the bubble formed by intradermal injection being approximately 20 mm), only part of the luciferase activity was collected. This may explain the lower apparent amount of luciferase obtained in the human situation compared to the mouse situation where the whole injection site (the whole ear) was used to measure luciferase activity.
Our experiments show for the first time that, in mice, the uptake of injected naked mRNA in the form of a globin UTR-stabilized molecule is efficient and strongly depends on the presence of calcium in the injection solution. We show evidence that this uptake is mediated by an active and saturable mechanism specific for nucleic acids. Several cell types in the mouse dermis are capable of taking up the foreign protein-coding RNA. We show that the uptake of exogenous mRNA can also be demonstrated in human skin. The capacity of cells to actively and qualitatively take up exogenous coding RNA is a surprising feature. It may be part of an understudied natural process involved in cell-to-cell communication. This pathway could be mediated by secretion and recapture of RNA by neighboring cells, as originally suggested by Benner.17 Such a process may be further regulated by the secreted RNases located between cells of the body. This hypothesis would explain why some extracellular growth factors such as angiogenins contain RNase activity. Whether RNA molecules can be short distance cell growth factors or differentiation signals and whether this capacity depends on the potential of RNA molecules to code for proteins is an intriguing concept that needs further investigation. Meanwhile, this process can be exploited for local and transient protein expression as described in the present work. We anticipate that our results, the infrastructure developed for the production of GMP-grade in vitro transcribed mRNA and the demonstration of in vivo mRNA transfer in human tissues, open the way to mRNA-based therapies.
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This work was supported by the Fritz-Bender Stiftung. JP was supported by the DFG Graduiertenkolleg ‘infektionsbiologie’ 685 of Tübingen.
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Probst, J., Weide, B., Scheel, B. et al. Spontaneous cellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent. Gene Ther 14, 1175–1180 (2007). https://doi.org/10.1038/sj.gt.3302964
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