commentary © The American Society of Gene & Cell Therapy

See page 1456 mRNA: Fulfilling the Promise of Gene injection of mice with 0.1 μg of pseu- douridine-containing EPO mRNA gave Therapy rise to EPO levels and reticulocyte values that are similar to what was achieved in Drew Weissman1 and Katalin Karikó2 the current study using 10 times more 4 doi:10.1038/mt.2015.138 GC-rich EPO mRNA. However, the most critical experiment is to unequivocally demonstrate a lack of immunogenicity of n vitro–transcribed mRNA (IVT mRNA) codon for each amino acid. When such the codon-optimized sequences. Although I is emerging as a new class of drug sequence-engineered mRNAs encoding Thess and colleagues reported no induc- that has the potential to play a role in firefly luciferase and erythropoietin (EPO) tion of tumor necrosis factor-α (TNF-α) that once was envisioned were transfected into cells, more protein and interleukin-6, these cytokines are not for DNA.1 Although first described as a was produced from the mRNA generated produced at sufficient levels when RNA is therapeutic in 1992,2 IVT mRNA’s immu- using conventional nucleotides compared formulated with TransIT, a commercially nogenicity prevented its development for to modified ones. The only condition in available polymer. It has been demon- protein replacement therapies. However, which the pseudouridine-modified IVT strated that whereas lipid-formulated this problem was recently solved by the mRNA was superior was when the IVT RNA induces TNF-α, TransIT-formulated introduction of modified nucleosides into mRNA contained the native GC-poor RNA primarily induces interferon-α (IFN- the IVT mRNA.3 In this issue of Molecular coding sequence. α).7 It has been reported that mice injected Therapy, Thess and colleagues report Using EPO-based hematopoiesis as with a positive control immunogenic an alternative method for generating an animal model system, they demon- RNA formulated in TransIT produce only nonimmunogenic IVT mRNA that avoids strated that injection of mice with 1 μg of IFN-α and not TNF-α or interleukin-6 the use of modified nucleosides.4 They sequence-engineered mRNA containing (ref. 6). Unfortunately, IFN-α was not demonstrate that sequence engineering uridines increases serum EPO levels and measured in any of the experiments. The of the mRNA and purifying it with high hematocrits to a higher level than the positive control RNA that induced all of performance liquid chromatography are corresponding pseudouridine-containing the measured inflammatory cytokines was sufficient to avoid immune activation and mRNA. Even repeated injections of unformulated (naked) and injected intra- to achieve high levels of translation of the 10 μg of sequence-engineered mRNA muscularly, whereas the test IVT mRNA encoded protein both in vitro and in vivo. (made with unmodified nucleotides) was TransIT-formulated and injected In previous studies using IVT mRNA was found to be nonimmunogenic, as intraperitoneally. in which all uridines were exchanged assessed by measuring inflammatory cyto- Future experiments will determine for pseudouridines—the most common kines, which remained at baseline levels whether the sequence-engineered RNA is naturally occurring modified nucleo- following treatment. Most importantly, nonimmunogenic or that it retains partial side—the mRNA was found to be very when pigs and macaques were injected immunogenicity. Nevertheless, it raises efficiently translated and nonimmuno- with lipid nanoparticle-formulated, an interesting question: why are GC-rich genic.5 Here, Thess and colleagues opted ­sequence-engineered EPO mRNA, the sequences less immunogenic? They are to alter the sequence composition of the authors observed significant increases prone to form stable double-stranded mRNA by incorporating the most GC-rich of reticulocyte numbers and hemato- structures and thus would be expected crits. Thus, IVT mRNA could achieve a to be more immunogenic. One expla- 1Division of Infectious Diseases, Department of physiologically relevant parameter in large nation could be that increasing the GC Medicine, University of Pennsylvania Perelman animals, thus opening potential opportu- content decreases the uridine (U) content School of Medicine, Philadelphia, Pennsylvania, nities to expand the therapeutic applica- of mRNA. Since U-rich RNA sequences 2 USA; BioNTech RNA Pharmaceuticals, , tion of IVT mRNA to treat other diseases. are known activators of several RNA Germany However, several important experi- sensors, including Toll-like receptor 7 Correspondence: , Department of Medicine, University of Pennsylvania ments remain. First, a side-by-side (ref. 8) and 8 (ref. 9), and RIG-I (ref. 10), Perelman School of Medicine, 522B Johnson comparison of the sequence-engineered it is possible that lowering the U content Pavilion, Philadelphia, Pennsylvania 19104, (GC-rich), uridine-containing mRNA vs. greatly reduces the immunogenicity of USA. E-mail: [email protected] or the native (GC-poor) mRNA containing the RNA. It is also conceivable that some Katalin Karikó, BioNTech RNA Pharmaceuticals, pseudouridine should be performed in pathogens exploit this strategy to avoid the An der Goldgrube 12, D-55131, Mainz, 6 Germany. E-mail: [email protected] vivo. It is curious that in earlier work, mammalian immune system. For example,

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the extremely high GC and therefore low sequence, as half of the codons, including 6. Karikó, K, Muramatsu, H, Keller, JM and Weissman, D (2012). Increased erythropoiesis in mice injected U content of the herpesvirus family and the start and the stop codons, must incor- with submicrogram quantities of pseudouridine- mycobacterial genomes might be critical porate uridines. containing mRNA encoding erythropoietin. Mol Ther for these pathogens to avoid activation of Considering the enormous potential 20: 948–953. the immune system. that IVT mRNA holds, we are looking 7. Karikó, K, Muramatsu, H, Ludwig, J and Weissman, D (2011). Generating the optimal mRNA for therapy: Thus, based on accumulated evidence, forward to learning about exciting results HPLC purification eliminates immune activation and it seems that to generate an optimal IVT as IVT mRNA enters clinical trials for improves translation of nucleoside-modified, protein- mRNA for protein therapy, one needs protein therapy. encoding mRNA. Nucleic Acids Res 39: e142. to reduce or completely eliminate its U 8. Diebold, SS, Massacrier, C, Akira, S, Paturel, C, Morel, REFERENCES Y and Reis e Sousa, C (2006). Nucleic acid agonists content without interfering with its trans- 1. Sahin, U, Kariko, K and Tureci, O (2014). mRNA- for Toll-like receptor 7 are defined by the pres- latability. This has been accomplished by based therapeutics––developing a new class of drugs. ence of uridine ribonucleotides. Eur J Immunol 36: (i) replacing all the uridines with pseu- Nat Rev Drug Discov 13: 759–780. 3256–3267. douridines, thus rendering the RNA 2. Jirikowski, GF, Sanna, PP, Maciejewski-Lenoir, D and 9. Heil, F, Hemmi, H, Hochrein, H, Ampenberger, F, Bloom, FE (1992). Reversal of diabetes insipidus 6 Kirschning, C, Akira, S et al. (2004). Species-specific nonimmunogenic; (ii) replacing 25% of in Brattleboro rats: intrahypothalamic injection of recognition of single-stranded RNA via Toll-like recep- the uridines with 2-thiouridines, resulting vasopressin mRNA. Science 255: 996–998. tor 7 and 8. Science 303: 1526–1529. in an mRNA with some residual immuno- 3. Karikó, K, Buckstein, M, Ni, H and Weissman, D 10. Saito, T, Owen, DM, Jiang, F, Marcotrigiano, J and genicity;11 or (iii) increasing the length of (2005). Suppression of RNA recognition by Toll-like Gale, M Jr (2008). Innate immunity induced by receptors: the impact of nucleoside modification composition-dependent RIG-I recognition of hepatitis the poly(A) tail so as to reduce the rela- and the evolutionary origin of RNA. Immunity 23: C virus RNA. Nature 454: 523–527. 165–175. tive U content or shielding the uridines in 11. Kormann, MS, Hasenpusch, G, Aneja, MK, Nica, the RNA sequence and generating RNA 4. Thess, A, Grund, S, Mui, BL, Hope, MJ, Baumhof, P, G, Flemmer, AW, Herber-Jonat, S et al. (2011). with reduced immune potential.12 Now, Fotin-Mleczek, M et al. (2015). Sequence-engineered Expression of therapeutic proteins after delivery of mRNA without chemical nucleoside modifications chemically modified mRNA in mice. Nat Biotechnol we can add a fourth strategy that involves enables an effective protein therapy in large animals. 29: 154–157. Mol Ther 23: 1456–1464 modifying the codons to render the 12. Koski, GK, Karikó, K, Xu, S, Weissman, D, Cohen, PA mRNA GC-rich, thus eliminating as many 5. Karikó, K, Muramatsu, H, Welsh, FA, Ludwig, J, Kato, and Czerniecki, BJ (2004). Cutting edge: innate im- uridines as possible leading to reduced H, Akira, S et al. (2008). Incorporation of pseudouri- mune system discriminates between RNA containing dine into mRNA yields superior nonimmunogenic bacterial versus eukaryotic structural features that or abolished immunogenicity. Obviously, vector with increased translational capacity and prime for high-level IL-12 secretion by dendritic cells. the level of U content will always depend biological stability. Mol Ther 16: 1833–1840. J Immunol 172: 3989–3993. on the amino acid content of the coding

See page 1475 Self-Destruct Genetic Switch to Safeguard iPS Cells Zoltán Ivics1 doi:10.1038/mt.2015.139

uicide gene approaches allow the context of induced pluripotent stem such as contaminating pluripotent S conditional elimination of gene- (iPS) cells.1 The authors of the study cells or their differentiated progeny modified cells and have been applied engineered human iPS cells to express with genomic/epigenomic abnormali- in cell therapy clinical trials. One such inducible caspase-9 (iC9) using lenti- ties having the potential to form tera- system, based on inducible apoptosis- viral vectors, and demonstrated rapid tomas following ­transplantation. mediated cell killing by caspase-9, CID-dependent apoptotic cell death Direct reprogramming of differen- which is activated by a nontoxic chem- in the parental iPS cells, in mesen- tiated somatic cells by gene transfer of ical inducer of dimerization (CID), has chymal stromal cells differentiated a small number of defined transcrip- now been successfully applied in the from the iPS cells in vitro, and in tion factors has been shown to yield teratomas generated in vivo following cells that are highly similar to embry- 1 Division of Medical , Paul Ehrlich pluripotent stem cell transplantation onic stem (ES) cells with respect to Institute, Langen, Germany in mice. Such a system is of para- gene expression, morphology, pluripo- Correspondence: Zoltán Ivics, Division of Medi- cal Biotechnology, Paul Ehrlich Institute, Paul mount importance for enhancing the tency, and capacity for in vitro differ- Ehrlich Str. 51-59, 63225 Langen, Germany. safety profiles of iPS cell–based prod- entiation.2 Due to their plasticity and E-mail: [email protected] ucts by elimination of unwanted cells, unlimited capacity for self-renewal,

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