mRNA-Based Genetic Reprogramming

The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient’s own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several “non-integrating” reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously “footprint-free,” most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic. The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient’s own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several “non-integrating” reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously “footprint-free,” most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic. Just over a decade has elapsed since the publication of Shinya Yamanaka’s groundbreaking work1Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Google Scholar showing that ordinary skin cells can be “reprogrammed” by the expression of a cocktail of transcription factors into induced pluripotent stem cells (iPSCs) capable of giving rise to any cell or tissue of the body. Already, the first regenerative therapies based on these cells, focused on age-related macular degeneration, ischemic heart disease, and Parkinson’s disease, have progressed to the stage of clinical trials.2Normile D. First-of-its-kind clinical trial will use reprogrammed adult stem cells to treat Parkinson’s.Science. 2018; (Published online July 30, 2018)https://doi.org/10.1126/science.aau9466Google Scholar The advent of cellular reprogramming holds out the tantalizing prospect it might be possible to turn a patient’s own cells into a limitless supply of physiologically rejuvenated, immunologically compatible stem cells that can be coaxed to become specialized cells, tissues, and organs for transplant back into the donor, enabling new treatments for a wide range of diseases and for the maladies of old age. Clearly, there is a long road ahead before this futuristic vision can be fully realized, with many technical, financial and regulatory obstacles to be overcome. A major hurdle to any clinical application of iPSCs made with Yamanaka’s original method was its dependence on integrating viral gene expression vectors to effect reprogramming, as the resulting heritable changes to cellular DNA would entail an unacceptable risk of tumorigenicity were the iPSCs or their differentiated progeny to be transferred into a human host. The goal of achieving “footprint-free” reprogramming obsessed the field for several years in the wake of Yamanaka’s breakthrough. This once-daunting challenge can now be considered a solved problem. One of the first compelling solutions presented relies on the sustained delivery of synthetic mRNA encoding Yamanaka’s reprogramming factors. Today, mRNA reprogramming vies with other well-established “non-integrating” methods, but it remains one of the most promising ways of making pluripotent stem cells for clinical use, based on the unambiguously transient character of the vector, the superior speed and efficiency of iPSC generation it provides, and the supple control it affords over reprogramming factor dosing, stoichiometry, and time course. This review surveys the emergence of mRNA reprogramming, looking at the roadblocks that had to be circumvented to bring it to fruition, improvements that have been made to the method since it was first described, areas where work remains to be done to address outstanding limitations, and progress toward the industrialization of this technique and its application in a clinical setting. It is appropriate here to recap the general character of cellular reprogramming, highlighting why techniques that alter the targeted cells’ DNA were employed in its early embodiments, and why this is a problem from a clinical perspective. Yamanaka asked if there is a way to take a differentiated cell such as a skin fibroblast and revert its gene expression profile back to an early embryonic state, so it becomes a blank slate with the potential to produce any of the cell lineages that make up the body. That this might be possible in principle is implicit in the fact that changes in gene expression rather than changes in DNA sequence underpin almost all known changes in cellular lineage and phenotype during development. More concretely, Sir John Gurdon’s work in the 1960s on somatic cell nuclear transfer revealed that the global expression profile of a nuclear genome isolated from a differentiated cell can be reset when it is transferred into an enucleated egg, presumably by factors such as proteins in the cytoplasm of the egg, allowing the formerly specialized genome to support development of the entire organism.3Gurdon J.B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles.J. Embryol. Exp. Morphol. 1962; 10: 622-640Google Scholar Subsequently, decades of research into the character of cellular differentiation have identified a class of nuclear proteins designated “transcription factors” that regulate the overall gene expression profile of every cell. In many cases, it has proved possible using classical genetic techniques to pinpoint specific transcription factors or small networks of cross-regulating factors that act as master regulators of cell fate. There were also a few published experiments, going back to the 1980s, in which transgene constructs were used to ectopically express master regulator factors within cells in vitro, resulting in broad phenotypic changes or putative cell-lineage conversions.4Graf T. Enver T. Forcing cells to change lineages.Nature. 2009; 462: 587-594Google Scholar Notably, transient expression of the muscle-specific transcription factor MyoD was shown to induce cultured fibroblasts to undergo a remarkable and enduring remodeling and conversion into multinucleate myotube-like cells.5Davis R.L. Weintraub H. Lassar A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts.Cell. 1987; 51: 987-1000Google Scholar The emergence of retroviral gene expression vectors as a powerful method for producing strong, heritable ectopic gene expression in cultured cells gave Yamanaka the means to conduct a large-scale screen for transcription factors that might trigger differentiated cells to activate genes associated with embryogenesis and pluripotency. Focusing on factors known to be associated with the embryonic state, Yamanaka transduced random combinations of transcription factor expression cassettes into cultured fibroblasts. He went on to identify the factors that had integrated into the genome of emergent colonies expressing selection markers indicative of embryonic reversion, then winnowed down these factors by a process of elimination to find especially potent factor combinations. He discovered that a cocktail of four factors (the so-called Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc) can reliably reprogram a small fraction of murine and human fibroblasts into cells that are almost indistinguishable from the embryonic stem cells (ESCs) cultured from early embryos.1Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Google Scholar, 6Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Google Scholar, 7Yamanaka S. Induced pluripotent stem cells: past, present, and future.Cell Stem Cell. 2012; 10: 678-684Google Scholar These rapidly growing, immortalized cells form compact colonies that can be picked, replated, and expanded, then differentiated under appropriate conditions to produce cells representative of all three embryonic germ layers (mesoderm, ectoderm, and endoderm), satisfying the basic criteria for pluripotency. The ability of integrating retroviral vectors to sustain gene expression at a high level over multiple rounds of cell division was crucial to the success of Yamanaka’s strategy. Even the modest levels of reprogramming efficiency seen in this early work, in which on the order of 0.01% of fibroblasts gave rise to iPSC clones, required weeks of continuous co-expression of the most potent factor combination. Expression of reprogramming factors from the transgenes does not have to be sustained indefinitely because they gradually activate a self-reinforcing “pluripotency network” of endogenous transcription factors that maintains the global embryonic gene expression pattern. In the retroviral system, native genome-defense pathways eventually methylate and silence the transgenes, which is crucial because their continued forced expression would inhibit differentiation of the iPSCs. Nonetheless, the presence of these transgenes in iPSC genomes entails serious risks from the standpoint of clinical application. In the first place, the silencing of the retroviral cassettes is not always an irreversible process. Sporadic reactivation of the Yamanaka transgenes, some of which are known to have oncogenic and immortalizing effects, could easily lead to the formation of tumors. Even the disruption of native DNA sequence brought about by the integration of retroviruses at random genomic sites bears some risk of dysregulating normal gene expression and, again, raises the specter of tumorigenicity. The scientific community swiftly embraced the potential for applying iPSCs to make almost any type of cell with any desired genetic background for use in developmental studies, disease modeling, and drug screening. For these types of research applications, the problem of the carryover of integrated transgenes from the reprogramming process is a marginal concern. Still, it was also recognized that novel reprogramming methods would have to be developed to overcome the problem of genome modification before iPSCs could become the basis for a new era of personalized regenerative medicine. The first steps in this direction involved the use of two well-established, non-integrating DNA-based gene expression vectors, plasmids and adenovirus.8Okita K. Nakagawa M. Hyenjong H. Ichisaka T. Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors.Science. 2008; 322: 949-953Google Scholar, 9Stadtfeld M. Nagaya M. Utikal K. Induced pluripotent stem cells without viral 322: Scholar to the gene expression when these vectors are into rapidly cells, these vectors gave iPSC than integrating and This was by the development of vectors which a of to of the with the K. K. S. induced pluripotent stem cells of and transgene 2009; Scholar Nonetheless, all of these DNA-based reprogramming vectors from the basic that and genome can at a need for before iPSCs made using these systems can be in a clinical K. S. as an integrating Scholar, H. DNA integration in 10: Scholar, M. M. H. M. M. and induced pluripotent stem cells for disease and Cell. Scholar the problem of genome modification was in by of reprogramming using Yamanaka factor proteins to cells rather than from vectors into the H. S. S. T. S. S. of induced pluripotent stem cells using Stem Cell. 2009; Scholar, D. S. Generation of human induced pluripotent stem cells by delivery of reprogramming Stem Cell. 2009; Scholar The factors used in this to their to the cell to of the by the cells. this from iPSC efficiency as a hurdle that has not been fully overcome in the years since and that technical of the D. S. T. mRNA is a for the delivery of proteins into the cell than with a Scholar There has since been of this hopes that a reprogramming might by developed to overcome the need for ectopic gene expression have to be realized, at in the human S. cell by small Cell. Scholar This is because of the of the genetic remodeling required to the of as by the fact that a factor cocktail is required to produce iPSC colonies with but a level of of the to have for a reprogramming that the of genomic modification are all These systems based on synthetic with a and synthetic from T. 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