mRNA Leapfrogs DNA to Show Promise for Therapeutic Gene Transfer (2012)

The notion of expressing genetically engineered therapeutic protein products in vivo from messenger RNA (mRNA)—notorious for its nuclease-sensitivity—is arguably counterintuitive. However, in an article appearing in an upcoming issue, Karikó and colleagues1Karikó K Muramatsu H Keller JM Weissman D Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin.Mol Ther. 2012; (in press)PubMed Google Scholar show that systemic administration of erythropoietin (EPO)-encoding mRNA to mice results in a sustained expression of EPO protein in the circulation that is accompanied by a significant increase in reticulocyte counts and hematocrits. The study shows that using mRNA as template for therapeutic gene transfer not only is efficient and capable of eliciting biologically significant effects but may prove in some instances superior to DNA-based therapy. Previous studies showing that intramuscular or intradermal injection of “naked” unmodified mRNA could elicit immune responses against the corresponding antigens and could even engender antitumor protective immunity in mice have gone largely unnoticed.2Conry RM LoBuglio AF Wright M Sumerel L Pike MJ Johanning F et al.Characterization of a messenger RNA polynucleotide vaccine vector.Cancer Res. 1995; 55: 1397-1400PubMed Google Scholar,3Granstein RD Ding W Ozawa H Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA.J Invest Dermatol. 2000; 114: 632-636Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Given the sensitivity of the immune system, a low efficiency of transfection that generated minute amounts of antigen was apparently sufficient to trigger an immune response in these studies. Indeed, mRNA-based genetic therapy has thus far been used primarily to stimulate protective (antitumor) immunity.4Kreiter S Diken M Selmi A Tureci O Sahin U Tumor vaccination using messenger RNA: prospects of a future therapy.Curr Opin Immunol. 2011; 23: 399-406Crossref PubMed Scopus (108) Google Scholar However, even for this application it is clear that significant improvements are needed. The introduction of specific motifs into the 5′ and 3′ untranslated regions of the mRNA, the use of a modified cap structure and a longer poly(A) tail, and the formation of complexes of the mRNA with polycations have significantly enhanced mRNA-based gene delivery to stimulate tumor immunity, such that the approach has reached phase I clinical trials in cancer patients.4Kreiter S Diken M Selmi A Tureci O Sahin U Tumor vaccination using messenger RNA: prospects of a future therapy.Curr Opin Immunol. 2011; 23: 399-406Crossref PubMed Scopus (108) Google Scholar Nevertheless, other applications will require higher levels of sustained expression of the mRNA-encoded protein to exert a biological or therapeutic effect. Examples include the treatment of blood coagulation deficiencies or anemia with clotting factors or EPO, respectively. The low efficiency of mRNA-based in vivo gene transfer, even with the modifications mentioned above, is due largely to its sensitivity to nucleases and hence its limited persistence in vivo. In principle, this limitation can be offset to some degree by increasing the administered dose. Cost considerations aside, as is often the case in cancer therapeutics, more is not necessarily better; the administered mRNA can elicit dose-dependent nonspecific immune responses with detrimental consequences.5Karikó K Ni H Capodici J Lamphier M Weissman D mRNA is an endogenous ligand for Toll-like receptor 3.J Biol Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (827) Google Scholar,6Diebold SS Kaisho T Hemmi H Akira S Reis e Sousa C Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2731) Google Scholar This is where Karikó and colleagues’ new study comes in. In 2008, Karikó et al. reported that replacing the uridines in an mRNA backbone with pseudouridines not only enhanced nuclease stability and translatability of the mRNA but also reduced off-target effects.7Karikó K Muramatsu H Welsh FA Ludwig J Kato H Akira S et al.Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.Mol Ther. 2008; 16: 1833-1840Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar The biological relevance of using such chemically modified mRNAs was demonstrated in a recent study by Kormann et al., who showed that intramuscular injection of EPO mRNA or aerosol administration of surfactant protein B (SP-B) into the lung of congenitally SP-B–deficient mice led to a significant increase in the average hematocrit and restoration of wild-type levels of SP-B, respectively.8Kormann MS Hasenpusch G Aneja MK Nica G Flemmer AW Herber-Jonat S et al.Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.Nat Biotechnol. 2011; 29: 154-157Crossref PubMed Scopus (514) Google Scholar In both studies, chemical modification of the mRNA backbone reduced but did not eliminate the nonspecific immune effects of the mRNA. Addressing this issue and more, Karikó et al. published a study last year showing that high-pressure liquid chromatography (HPLC) purification of the in vitro–transcribed mRNA further enhanced its translatability and eliminated the residual off-target effects,9Karikó K Muramatsu H Ludwig J Weissman D Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA.Nucleic Acids Res. 2011; 39: e142Crossref PubMed Scopus (420) Google Scholar representing a prime example of the devil being in the details. In their study, Karikó and colleagues put the mRNA approach to a stringent test. They found that a single intraperitoneal administration of a low dose (100 ng) of the chemically modified, HPLC-purified mRNA led to significant and sustained (up to 4 days) EPO expression in the circulation and that repeated administrations were accompanied by clear EPO-associated biological effects in the treated mice: increased reticulocyte counts and hematocrits. Notably, administration of recombinant EPO protein, the standard of care in treating anemia, was not nearly as effective; it exhibited a characteristic short circulation half-life of 6 hours and required large amounts of protein to elicit a comparable increase in reticulocytes. In a pilot experiment, administration of EPO mRNA to rhesus macaques also led to significantly increased serum levels of EPO. Importantly, administration of even high doses of the chemically modified HPLC-purified mRNA to mice did not elicit nonspecific immune responses, in stark contrast to the effects of unmodified mRNA, which was also significantly less efficacious. Administration of proteins often elicits neutralizing antibodies, which in some instances are associated with serious adverse immune reactions, and EPO protein therapy is a case in point.10Casadevall N Nataf J Viron B Kolta A Kiladjian JJ Martin-Dupont P et al.Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin.N Engl J Med. 2002; 346: 469-475Crossref PubMed Scopus (1113) Google Scholar Tantalizingly, administration of EPO-encoding mRNA to mice did not elicit antibodies specific to EPO, suggesting that the mRNA approach will be also more effective and safer in this regard. It is not immediately clear why the mRNA-translated EPO protein was not immunogenic. Is it the continuous production of low levels of the mRNA-encoded protein vs. the high-dose yet short-lived recombinant protein? Is it a general distinction between administered recombinant protein and mRNA-expressed product, or is it unique to EPO? DNA plasmids and viral vectors can elicit nonspecific immune responses with detrimental consequences11Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4742) Google Scholar and can potentially integrate into the host genome. The mRNA approach is clearly more efficacious than protein administration from the standpoint of sustained production of biologically active products. How it compares to plasmid DNA or viral vectors is not clear, but it might be a moot issue given the considerations mentioned above. mRNA's safety profile is superior to that of protein, plasmid DNA, or viral vectors. It does not integrate into the host genome during its transient presence; the chemically modified HPLC-purified formulation is nonimmunogenic and does not appear to stimulate neutralizing antibodies. No less important, clinical-grade mRNA can be generated by a relatively cost-effective process—perhaps not as cost-effective as for plasmid DNA but much less costly than production of viral vectors or protein products. Notwithstanding these attractive features, the mRNA approach will be less useful for applications that require extended or lifelong expression of the therapeutic product. The choice of vector will ultimately be determined by weighing the pros and cons of the transient mRNA approach vs. stable gene transfer methods using viral vectors. It will be important to extend these observations to other clinically relevant models in mice (e.g., immunotherapy, clotting deficiencies and surfactant protein-B deficiency) and to confirm the observations in the nonhuman primate model. It is safe to assume that the bar will be much higher in human patients than in animal models, from the standpoints of both effectiveness and safety. Arguably, additional improvements of efficacy, safety, and feasibility will probably be necessary. The primary role of animal modeling is to screen among various options to choose those more likely to perform better in human patients, not to inform whether a particular treatment will exhibit a therapeutic impact in patients and of what magnitude. The report by Karikó and colleagues has helped (me) make a choice, for it is indeed hard to be left unimpressed. Unthinkable a mere decade ago, the pioneering studies from the laboratories of Weissman, Sahin, and a few other investigators have brought closer to reality the concept of using RNA administered directly to patients to express therapeutic proteins and thus even to leapfrog DNA-based approaches for certain applications.