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Sequence-Engineered Mrna Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

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Sequence-Engineered Mrna Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals © The American Society of Gene & Cell Therapy original article original article MTOpen Protein Therapy With Sequence-engineered mRNA © The American Society of Gene & Cell Therapy Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals Andreas Thess1, Stefanie Grund1, Barbara L Mui2, Michael J Hope2, Patrick Baumhof1, Mariola Fotin-Mleczek1 and Thomas Schlake1 1CureVac GmbH, Tübingen, Germany; 2Acuitas Therapeutics, Vancouver, British Columbia, Canada Being a transient carrier of genetic information, mRNA machinery. Whereas this feature could threaten the use of mRNA could be a versatile, flexible, and safe means for protein for protein therapies, immunostimulation may be beneficial for therapies. While recent findings highlight the enormous vaccines composed of antigen-encoding mRNA. Indeed, immuni- therapeutic potential of mRNA, evidence that mRNA- zation with mRNAs coding for cancer antigens was successfully based protein therapies are feasible beyond small animals established and has already entered clinical trials.15–19 such as mice is still lacking. Previous studies imply that Analysis of various RNA preparations from bacteria and mRNA therapeutics require chemical nucleoside modifi- eukaryotic cells revealed significant differences as to their immu- cations to obtain sufficient protein expression and avoid nostimulatory properties.20 Obviously, cytokine secretion by RNA- activation of the innate immune system. Here we show transfected cells strongly correlated with the extent of nucleoside that chemically unmodified mRNA can achieve those modifications. Transferring this concept to in vitro transcribed goals as well by applying sequence-engineered mol- mRNA, for instance, pseudouridine-containing mRNAs reduced ecules. Using erythropoietin (EPO) driven production of activation of known RNA sensors substantially.20–22 Although pseu- red blood cells as the biological model, engineered Epo douridine is primarily found in tRNA, rRNA, and small nuclear mRNA elicited meaningful physiological responses from RNAs, pseudouridine-containing mRNAs were still translated and 22,23 mice to nonhuman primates. Even in pigs of about 20 kg produced even more protein compared to unmodified mRNA. in weight, a single adequate dose of engineered mRNA Accordingly, mRNA harboring modified nucleosides was sug- encapsulated in lipid nanoparticles (LNPs) induced high gested as means of choice for protein expression via mRNA. systemic Epo levels and strong physiological effects. Our Using enhanced green fluorescent protein mRNA, Rossi and results demonstrate that sequence-engineered mRNA has colleagues confirmed that nucleoside modifications can strongly 24 the potential to revolutionize human protein therapies. enhance protein expression and suppress cytokine secretion. In contrast to previous work, they applied a combination of pseu- Received 30 January 2015; accepted 27 May 2015; advance online douridine and 5-methyl-cytidine which outperformed each single publication 30 June 2015. doi:10.1038/mt.2015.103 modification. As in earlier studies, unmodified nucleotides were INTRODUCTION completely replaced by their modified counterparts. With such Messenger RNA is an intermediate carrier of genetic information mRNAs, the authors succeeded in reprogramming human cells that is used by organisms as template for protein expression. Thus, to pluripotency. The same type of mRNA modification allowed 30January2015 mRNA may also serve as a tool for the expression of proteins of vascular regeneration after myocardial infarction in mice by local interest by introducing exogenous molecules into target cells. This expression of VEGF.25 However, different groups apparently favor 27May2015 concept was first put to the test in the early 1970s by microinjecting different modified nucleosides.26 Moreover, according to the find- RNA preparations into Xenopus oocytes, demonstrating the synthe- ings of Kormann et al.26, partially modified mRNA provides the 10.1038/mt.2015.103 sis of RNA-encoded proteins.1,2 Meanwhile, loading of dendritic cells best combination of high expression and low immunostimulation with antigen-encoding mRNA, described for the first time by Gilboa to achieve therapeutic effects in mice. 3 Molecular Therapy and colleagues, became a widely applied immunological approach. Two independent studies on erythropoietin-encoding mRNA In the early 1990s, first studies demonstrated that exogenous found a superiority of nucleoside-modified compared to unmodi- 26,27 23 mRNA can be used to direct protein expression in vivo. After local fied mRNA. Whereas unmodified Epo mRNA was immu- injection of various mRNAs into mouse muscle led to detectable nostimulatory and failed to induce sufficient protein expression protein levels, the treatment of vasopressin-deficient rats provided in mice, a few micrograms of nucleoside-modified mRNA were 9 first evidence for mRNA as a potential therapeutic.4–6 However, enough to achieve detectable protein expression and physi- several RNA structural features have been described as immuno- ological effects upon direct administration to murine muscle.26 1456 stimulatory due to interactions with various RNA sensors such as Complexation with TransIT, enabling intraperitoneal injections, Toll-like receptors, RIG-I, and PKR.7–14 Activation of these receptors reduced the dose threshold of nucleoside-modified mRNA to just a 1464 provides a danger signal which may interfere with the translational few nanogram in mice.27 Repeated treatments sustainably increased 00sep2015 Correspondence: Thomas Schlake, CureVac GmbH, Paul-Ehrlich-Str. 15, 72076 Tübingen, Germany. E-mail: [email protected] 30June2015 1456 www.moleculartherapy.org vol. 23 no. 9, 1456–1464 sep. 2015 © The American Society of Gene & Cell Therapy Protein Therapy With Sequence-engineered mRNA the hematocrit without any signs of immunostimulation, assessed a 108 27 5 ng via interferon-α secretion and anti-EPO antibody generation. In 50 ng 7 addition, a weight-adjusted mRNA dose enhanced serum levels of 10 500 ng 27 erythropoietin in Rhesus macaques. Unfortunately, physiological 106 responses in monkeys were not reported in that study. These data raised the question whether nucleoside-modifica- 105 tion is an inevitable prerequisite for the development of mRNA- 104 based protein therapies. Here, using sequence-engineered mRNA, 3 we demonstrate that unmodified mRNA is fully suited for use in PpLuc (RLU) mean, SD 10 protein therapies. Engineered but chemically unmodified mRNA 102 coding for erythropoietin achieved high systemic protein levels Unmod ψψ + 5 mC and strong physiological responses in vivo. Moreover, immunos- b 4 timulation of that mRNA was at background levels. In combination 10 5 ng with an appropriate formulation, e.g., LNPs designed for systemic 50 ng 3 500 ng administration,28 sequence-engineered Epo mRNA elicited mean- 10 ingful physiological responses even in domestic pigs with a weight 102 of 20 kg. Thus, strong evidence is provided that mRNA-based pro- tein therapies are feasible in large animals, paving the way for the 101 development of a novel class of human therapeutics. 0 Epo (ng/ml) mean, SD 10 RESULTS Potent protein expression in vitro with unmodified 10−1 mRNA Unmod ψψ + 5 mC c Numerous studies from the early 1990s onwards advocate chemi- 8 10 5 ng cally unmodified mRNA as a suitable and potent means to induce 50 ng 7 antigen-specific immune responses,19,29–34 thereby indicating that 10 500 ng such nucleic acids do give rise to expression of encoded proteins 106 upon in vivo delivery. However, it is widely assumed and published 5 that unmodified mRNA is improper for therapeutic purposes due 10 to usually higher protein expression demands compared to vacci- 104 nation and potentially detrimental immunostimulation. However, 3 PpLuc (RLU) mean, SD 10 the finding that unmodified mRNA gives only very little protein expression contrasts with our experience with sequence-engineered 102 nucleic acids.35 Hence, we set out to test the notion of unmodified Basic (unmod) Basic (ψ) mRNA being appropriate for the expression of therapeutic proteins. Figure 1 Sequence-engineered mRNA yields high protein expres- First, we designed a firefly luciferase-encoding mRNA apply- sion in vitro. (a) Engineered, unmodified luciferase mRNA outperforms ing a sequence optimization approach which adapts the codon its nucleoside-modified counterparts. HeLa cells were lipofected in usage and selects the most appropriate regulatory sequences such triplicate with the indicated amounts of a luciferase-encoding mRNA that was either unmodified or harbored the given nucleoside modifi- as 5’ and 3’ untranslated regions in a target and application spe- cations. Luciferase expression was quantified 24 hours after transfec- cific manner. To test for potential additive effects, we also pro- tion. (b) Engineered but unmodified mouse Epo mRNA is superior to duced nucleoside-modified counterparts of the final sequence. nucleoside-modified variants. HeLa cells were lipofected in triplicate with Notably, in vitro protein expression revealed very high luciferase different amounts of an erythropoietin-encoding mRNA that was either unmodified or modified by incorporation of the indicated nucleosides. activity with the unmodified mRNA, while its nucleoside-mod- Erythropoietin levels in the supernatant were quantified 24 hours after ified counterparts gave rise to substantially lower protein levels transfection. (c) Pseudouridine
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