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Efficient Delivery and Functional Expression of Transfected Modified mRNA in Human Embryonic Stem Cell-derived Retinal Pigmented Epithelial Cells

Open AccessPublished:January 02, 2015DOI:https://doi.org/10.1074/jbc.M114.618835
      Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.

      Background

      In vitro-produced retinal pigmented epithelial (RPE) cells represent a novel source for retinal degenerative disease healing.

      Results

      mRNA transfection outperformed plasmid transfection in cellular uptake. Modified-mRNA displayed negligible immune activation and functional protein expression.

      Conclusion

      Modified mRNA transfection can be used efficiently for the engineering of RPE cells.

      Significance

      The modified mRNA transfection technique offers new venues for the treatment of RPE-related diseases.

      Introduction

      The retinal pigmented epithelium (RPE)
      The abbreviations used are: RPE
      retinal pigmented epithelial/epithelium
      hESC
      human embryonic stem cell
      hiPSC
      human induced pluripotent stem cell
      MITF
      microphthalmia-associated transcription factor
      TTR
      transthyretin
      qRT-PCR
      quantitative RT-PCR
      PI
      propidium iodide
      mfi
      mean fluorescence intensity
      unmod
      unmodified
      mod
      modified.
      is a layer of pigmented cells located between the choroid and the photoreceptors, where it serves as a part of the barrier between the bloodstream and the retina. This monolayer of cells operates in multiple ways, all of which are crucial for visual function, including light absorption, recycling of retinoids, epithelial transport, secretion of proteins, spatial ion buffering, phagocytosis of the outer segments of the photoreceptors, and immune regulation (
      • Strauss O.
      The retinal pigment epithelium in visual function.
      ).
      Dysfunction, degeneration, and loss of RPE cells are major characteristics of many retinal diseases, such as Stargardt disease, Best disease, subtypes of retinitis pigmentosa, proliferative vitreoretinopathy, and age-related macular degeneration, which all lead to gradual loss of visual acuity and, eventually, in many cases, to blindness (
      • Sparrow J.R.
      • Hicks D.
      • Hamel C.P.
      The retinal pigment epithelium in health and disease.
      ). A variety of therapeutic approaches to delay or repair retinal degeneration is under development, including cell-based and gene replacement therapies (
      • Sparrow J.R.
      • Hicks D.
      • Hamel C.P.
      The retinal pigment epithelium in health and disease.
      ,
      • Tucker B.A.
      • Mullins R.F.
      • Stone E.M.
      Stem cells for investigation and treatment of inherited retinal disease.
      ). Clinical trials involving the transplantation of intact sheets or single-cell suspensions of primary RPE cells have been carried out with mixed results (
      • Falkner-Radler C.I.
      • Krebs I.
      • Glittenberg C.
      • Povazay B.
      • Drexler W.
      • Graf A.
      • Binder S.
      Human retinal pigment epithelium (RPE) transplantation: outcome after autologous RPE-choroid sheet and RPE cell-suspension in a randomised clinical study.
      ).
      Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), as well as some somatic cell types such as mesenchymal stem cells, are all attractive cell sources for transplantation. Several reports have demonstrated that both hESCs and hiPSCs can differentiate in vitro into a functional monolayer of pigmented RPE-like cells (
      • Idelson M.
      • Alper R.
      • Obolensky A.
      • Ben-Shushan E.
      • Hemo I.
      • Yachimovich-Cohen N.
      • Khaner H.
      • Smith Y.
      • Wiser O.
      • Gropp M.
      • Cohen M.A.
      • Even-Ram S.
      • Berman-Zaken Y.
      • Matzrafi L.
      • Rechavi G.
      • Banin E.
      • Reubinoff B.
      Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.
      • Klimanskaya I.
      • Hipp J.
      • Rezai K.A.
      • West M.
      • Atala A.
      • Lanza R.
      Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.
      ,
      • Buchholz D.E.
      • Hikita S.T.
      • Rowland T.J.
      • Friedrich A.M.
      • Hinman C.R.
      • Johnson L.V.
      • Clegg D.O.
      Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells.
      • Carr A.J.
      • Vugler A.A.
      • Hikita S.T.
      • Lawrence J.M.
      • Gias C.
      • Chen L.L.
      • Buchholz D.E.
      • Ahmado A.
      • Semo M.
      • Smart M.J.
      • Hasan S.
      • da Cruz L.
      • Johnson L.V.
      • Clegg D.O.
      • Coffey P.J.
      Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.
      ) and that human embryonic stem cell-derived RPE can restore vision in the retinal dystrophy rat model (
      • Lund R.D.
      • Wang S.
      • Klimanskaya I.
      • Holmes T.
      • Ramos-Kelsey R.
      • Lu B.
      • Girman S.
      • Bischoff N.
      • Sauvé Y.
      • Lanza R.
      Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.
      ). In addition, by using a mixture of transcription factors, fibroblasts can be directed to trans-differentiate toward RPE-like cells (
      • Zhang K.
      • Liu G.H.
      • Yi F.
      • Montserrat N.
      • Hishida T.
      • Esteban C.R.
      • Izpisua Belmonte J.C.
      Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors.
      ). Recently, the first description of transplanted human ES cell-derived RPE cells into human patients was reported (
      • Schwartz S.D.
      • Hubschman J.P.
      • Heilwell G.
      • Franco-Cardenas V.
      • Pan C.K.
      • Ostrick R.M.
      • Mickunas E.
      • Gay R.
      • Klimanskaya I.
      • Lanza R.
      Embryonic stem cell trials for macular degeneration: a preliminary report.
      ), and, in Japan, a pilot clinical study on transplantation of autologous hiPSC-RPE cells has been initiated. Despite the great potential of these cells for future treatment of retinal degeneration, there are still some challenges regarding the degree of cell survival, immune rejection, and efficiency of engraftment. In addition, functional and molecular studies have shown that human ES cell- and hiPSC-derived RPE cells possess specific properties that are absent from currently available cell lines, such as ARPE-19, which make them useful for in vitro disease modeling or drug screening (
      • Klimanskaya I.
      • Hipp J.
      • Rezai K.A.
      • West M.
      • Atala A.
      • Lanza R.
      Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.
      ,
      • Melville H.
      • Carpiniello M.
      • Hollis K.
      • Staffaroni A.
      • Golestaneh N.
      Stem cells: a new paradigm for disease modeling and developing therapies for age-related macular degeneration.
      ,
      • Strunnikova N.V.
      • Maminishkis A.
      • Barb J.J.
      • Wang F.
      • Zhi C.
      • Sergeev Y.
      • Chen W.
      • Edwards A.O.
      • Stambolian D.
      • Abecasis G.
      • Swaroop A.
      • Munson P.J.
      • Miller S.S.
      Transcriptome analysis and molecular signature of human retinal pigment epithelium.
      ). Regardless of the application of hESC RPE or hiPSC RPE, a safe, flexible, and efficient gene delivery system is still needed. However, optimal gene delivery systems for RPE cells are limited.
      The use of synthetic mRNA as a gene delivery technique holds several benefits over classical DNA-based methods. Nevertheless, because of the relatively low half-life and the strong immunogenicity of conventional mRNA, the clinical application of this technique has been delayed. However, recent groundbreaking advances have established that replacing uridine and cytidine with pseudouridine and 5-methylcytidine, respectively, allows synthetic mRNA to bypass the cellular innate immune response (
      • Karikó K.
      • Buckstein M.
      • Ni H.
      • Weissman D.
      Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.
      ), which, in turn, opens the door to DNA-free cellular engineering strategies that would avoid any risks of genomic recombination or insertional mutagenesis. Because the transfected mRNA only has to reach the cytoplasm to achieve protein expression, the efficiency of transfection is also relatively high for cells that are considered to be difficult to transfect, such as postmitotic cells, by classical DNA-based delivery methods (because DNA must cross the nuclear envelope in addition to the plasma membrane). Modified mRNA has also been reported to have a higher translational capacity and stability than unmodified mRNA (
      • Karikó K.
      • Muramatsu H.
      • Welsh F.A.
      • Ludwig J.
      • Kato H.
      • Akira S.
      • Weissman D.
      Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.
      ,
      • Anderson B.R.
      • Muramatsu H.
      • Jha B.K.
      • Silverman R.H.
      • Weissman D.
      • Karikó K.
      Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNase L.
      ). Since its discovery, transfection of modified mRNA has been applied successfully in different research areas, including disease treatment (
      • Wang Y.
      • Su H.H.
      • Yang Y.
      • Hu Y.
      • Zhang L.
      • Blancafort P.
      • Huang L.
      Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy.
      ,
      • Kormann M.S.
      • Hasenpusch G.
      • Aneja M.K.
      • Nica G.
      • Flemmer A.W.
      • Herber-Jonat S.
      • Huppmann M.
      • Mays L.E.
      • Illenyi M.
      • Schams A.
      • Griese M.
      • Bittmann I.
      • Handgretinger R.
      • Hartl D.
      • Rosenecker J.
      • Rudolph C.
      Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.
      ,
      • Mays L.E.
      • Ammon-Treiber S.
      • Mothes B.
      • Alkhaled M.
      • Rottenberger J.
      • Müller-Hermelink E.S.
      • Grimm M.
      • Mezger M.
      • Beer-Hammer S.
      • von Stebut E.
      • Rieber N.
      • Nürnberg B.
      • Schwab M.
      • Handgretinger R.
      • Idzko M.
      • Hartl D.
      • Kormann M.S.
      Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism.
      ), vaccination (
      • Petsch B.
      • Schnee M.
      • Vogel A.B.
      • Lange E.
      • Hoffmann B.
      • Voss D.
      • Schlake T.
      • Thess A.
      • Kallen K.J.
      • Stitz L.
      • Kramps T.
      Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection.
      ), and regenerative medicine (
      • Lui K.O.
      • Zangi L.
      • Silva E.A.
      • Bu L.
      • Sahara M.
      • Li R.A.
      • Mooney D.J.
      • Chien K.R.
      Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.
      ,
      • Warren L.
      • Manos P.D.
      • Ahfeldt T.
      • Loh Y.H.
      • Li H.
      • Lau F.
      • Ebina W.
      • Mandal P.K.
      • Smith Z.D.
      • Meissner A.
      • Daley G.Q.
      • Brack A.S.
      • Collins J.J.
      • Cowan C.
      • Schlaeger T.M.
      • Rossi D.J.
      Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.
      ,
      • Zangi L.
      • Lui K.O.
      • von Gise A.
      • Ma Q.
      • Ebina W.
      • Ptaszek L.M.
      • Später D.
      • Xu H.
      • Tabebordbar M.
      • Gorbatov R.
      • Sena B.
      • Nahrendorf M.
      • Briscoe D.M.
      • Li R.A.
      • Wagers A.J.
      • Rossi D.J.
      • Pu W.T.
      • Chien K.R.
      Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.
      ).
      Here we demonstrate that synthetic unmodified mRNA, as well as modified mRNA, can be delivered efficiently into RPE cells independently of differentiation stage or confluence. However, administration of unmodified mRNA induces nuclear translocation of the immunogenic transcription factors IRF3 and p65/RelA and, consequently, a strong activation of their target genes, IFNβ and TNFα. In contrast, in modified mRNA-transfected cells, nuclear localization of IRF3 or p65/RelA is absent, showing minimal activation of TNFα and IFNβ. Similarly, the transcriptional activator MITF is more biologically active when expressed from modified mRNA than unmodified mRNA, as evidenced by the higher activation of its target genes. Therefore, synthetic modified mRNA offers an unprecedented opportunity for the study of RPE-related diseases and, potentially, to improve the therapeutic outcomes of degenerated RPE, including cell-based and gene-based therapies.

      DISCUSSION

      Development of a safe and efficient viral-free gene delivery system is one of the major challenges for gene-based therapies of the retina. Despite recent improvements in plasmid DNA delivery, regarding efficiency and viability, the efficiency of transfection is still considered to be low or moderate (seldom over 50%) (
      • Puras G.
      • Mashal M.
      • Zárate J.
      • Agirre M.
      • Ojeda E.
      • Grijalvo S.
      • Eritja R.
      • Diaz-Tahoces A.
      • Martínez Navarrete G.
      • Avilés-Trigueros M.
      • Fernández E.
      • Pedraz J.L.
      A novel cationic niosome formulation for gene delivery to the retina.
      ,
      • Sunshine J.C.
      • Sunshine S.B.
      • Bhutto I.
      • Handa J.T.
      • Green J.J.
      Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo.
      ). Moreover, the most successful in vitro plasmid DNA gene delivery studies were on RPE-derived cell lines, which are easier to transfect than primary RPE cells (
      • Vercauteren D.
      • Piest M.
      • van der Aa L.J.
      • Al Soraj M.
      • Jones A.T.
      • Engbersen J.F.
      • De Smedt S.C.
      • Braeckmans K.
      Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes.
      ). In this report, we used hESC-derived RPE cells, because they are considered to be much more similar to primary RPE than RPE cell lines (
      • Klimanskaya I.
      • Hipp J.
      • Rezai K.A.
      • West M.
      • Atala A.
      • Lanza R.
      Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.
      ), to investigate the utility of using synthetic mRNA as a novel gene delivery approach for transient protein expression in primary RPE cells. hESC-RPE cells were generated using a protocol reported previously with minor modifications (
      • Idelson M.
      • Alper R.
      • Obolensky A.
      • Ben-Shushan E.
      • Hemo I.
      • Yachimovich-Cohen N.
      • Khaner H.
      • Smith Y.
      • Wiser O.
      • Gropp M.
      • Cohen M.A.
      • Even-Ram S.
      • Berman-Zaken Y.
      • Matzrafi L.
      • Rechavi G.
      • Banin E.
      • Reubinoff B.
      Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.
      ). To ensure the highest purity and maturity of our generated hESC-RPE cultures, we selected and expanded the cultures for two rounds of passage before characterizing the RPE cell cultures (
      • Singh R.
      • Phillips M.J.
      • Kuai D.
      • Meyer J.
      • Martin J.M.
      • Smith M.A.
      • Perez E.T.
      • Shen W.
      • Wallace K.A.
      • Capowski E.E.
      • Wright L.S.
      • Gamm D.M.
      Functional analysis of serially expanded human iPS cell-derived RPE cultures.
      ). Morphological and molecular analysis of the cells showed typical RPE features such as tight junctions, melanin granules, apical microvilli, and expression of RPE gene transcripts and proteins important for many of the typical RPE functions. In many retinal diseases, including age-related macular degeneration and proliferative vitroretinopathy, RPE cells lose their epithelial phenotype and cell-to-cell contact and become proliferative, motile fibroblast-like cells (
      • Saika S.
      • Yamanaka O.
      • Flanders K.C.
      • Okada Y.
      • Miyamoto T.
      • Sumioka T.
      • Shirai K.
      • Kitano A.
      • Miyazaki K.
      • Tanaka S.
      • Ikeda K.
      Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis.
      ). A similar process also occurs when subculturing the RPE cells in vitro (
      • Klimanskaya I.
      • Hipp J.
      • Rezai K.A.
      • West M.
      • Atala A.
      • Lanza R.
      Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.
      ,
      • Buchholz D.E.
      • Hikita S.T.
      • Rowland T.J.
      • Friedrich A.M.
      • Hinman C.R.
      • Johnson L.V.
      • Clegg D.O.
      Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells.
      ,
      • Aronson J.F.
      Human retinal pigment cell culture.
      ). However, when confluence is reestablished, the cells regain their classical pigmented polygonal RPE morphology. Consistent with similar studies on primary RPE cells, RPE cell lines, as well as iPSC- and ES cell-derived RPE cells, we found the RPE-specific genes to be drastically down-regulated in the subconfluent cell culture (
      • Liu Y.
      • Ye F.
      • Li Q.
      • Tamiya S.
      • Darling D.S.
      • Kaplan H.J.
      • Dean D.C.
      Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology.
      ,
      • Adijanto J.
      • Castorino J.J.
      • Wang Z.X.
      • Maminishkis A.
      • Grunwald G.B.
      • Philp N.J.
      Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.
      ). A time course study of RPE cell reestablishment showed reexpression of RPE-specific transcripts as the cells became confluent. These observations were coincident with the acquisition of the typical polygonal cell shape and the re-establishment of pigmentation. The underlying mechanisms for the loss of RPE phenotype after cell-cell dissociation are not fully understood, but studies in mouse primary RPE cells and RPE cell lines suggest that loss of contact inhibition induces activation of canonical Wnt and Smad/ZEB (Zinc finger E-box-binding homeobox) signaling and subsequent activation of an epithelial-to-mesenchymal transition and unlocking of the mitotic block (
      • Liu Y.
      • Ye F.
      • Li Q.
      • Tamiya S.
      • Darling D.S.
      • Kaplan H.J.
      • Dean D.C.
      Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology.
      ,
      • Chen H.C.
      • Zhu Y.T.
      • Chen S.Y.
      • Tseng S.C.
      Selective activation of p120ctn-Kaiso signaling to unlock contact inhibition of ARPE-19 cells without epithelial-mesenchymal transition.
      ,
      • Liu Y.
      • Xin Y.
      • Ye F.
      • Wang W.
      • Lu Q.
      • Kaplan H.J.
      • Dean D.C.
      Taz-tead1 links cell-cell contact to zeb1 expression, proliferation, and dedifferentiation in retinal pigment epithelial cells.
      ). Moreover, it has been shown that ZEB can bind to the MITF promoter and, thereby, repress its transcription, which, in turn, would lead to down-regulation of MITF target genes, including many RPE-specific genes (
      • Liu Y.
      • Ye F.
      • Li Q.
      • Tamiya S.
      • Darling D.S.
      • Kaplan H.J.
      • Dean D.C.
      Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology.
      ,
      • Liu Y.
      • Xin Y.
      • Ye F.
      • Wang W.
      • Lu Q.
      • Kaplan H.J.
      • Dean D.C.
      Taz-tead1 links cell-cell contact to zeb1 expression, proliferation, and dedifferentiation in retinal pigment epithelial cells.
      ).
      Our analysis of the two transcription factors essential for RPE maintenance and function, Otx2 and Mitf, showed a moderate down-regulation of both transcripts in the subconfluent culture (4 days after passage). However, when analyzing these protein levels in 4-day subconfluent cells and 25-day cultured postconfluent RPE cells, we found that the OTX2 protein was undetectable, whereas the MITF protein could be detected readily (whereas, in 25-day postconfluent cells, both MITF and OTX2 were detectable). Because many of the analyzed RPE-related genes that were down-regulated (such as RPE65, CRALBP, and TTR) have been reported recently to be direct targets of OTX2 (
      • Masuda T.
      • Wahlin K.
      • Wan J.
      • Hu J.
      • Maruotti J.
      • Yang X.
      • Iacovelli J.
      • Wolkow N.
      • Kist R.
      • Dunaief J.L.
      • Qian J.
      • Zack D.J.
      • Esumi N.
      Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium.
      ,
      • Housset M.
      • Samuel A.
      • Ettaiche M.
      • Bemelmans A.
      • Béby F.
      • Billon N.
      • Lamonerie T.
      Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration.
      ), we speculate that, together with MITF, OTX2 may be responsible for the down-regulation of the RPE-specific genes after cell subculture.
      Despite the great interest in hESC-RPE and hiPSC RPE cells as sources for cell therapy and in vitro disease modeling, no studies of gene delivery of these cells have, to our knowledge, been reported. We have shown that, as is the case for RPE cells, hESC-RPE cells are very difficult to transfect with plasmid DNA complexed with commercial transfection reagent (FuGENE 6). The highest efficiency of transfection with plasmid DNA was achieved on subconfluent RPE cells with an efficiency of 17%, and this decreased as the hESC-RPE cells progressed into confluence. When the cells reached a polygonal morphology, less than 1% of the cell culture was positively transfected. In contrast, synthetic unmodified and modified (UTP and CTP substituted with pseudo-UTP and 5-methyl-CTP) mRNAs were delivered efficiently into subconfluent, early confluent, and postconfluent (polygonal) RPE cells. Mean fluorescence intensities were approximately equally high in cells transfected with plasmid, unmod, and mod mRNA-GFP. This is in contrast to some reports where modified mRNA is claimed to have a higher translation efficiency than unmodified mRNA (
      • Karikó K.
      • Muramatsu H.
      • Welsh F.A.
      • Ludwig J.
      • Kato H.
      • Akira S.
      • Weissman D.
      Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.
      ,
      • Warren L.
      • Manos P.D.
      • Ahfeldt T.
      • Loh Y.H.
      • Li H.
      • Lau F.
      • Ebina W.
      • Mandal P.K.
      • Smith Z.D.
      • Meissner A.
      • Daley G.Q.
      • Brack A.S.
      • Collins J.J.
      • Cowan C.
      • Schlaeger T.M.
      • Rossi D.J.
      Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.
      ), which should result in higher fluorescence in mod mRNA-transfected cells. The reason for this discrepancy is not clear, but because we observed a similar results in other cells (keratinocytes, HEK293T, and mesenchymal stem cells derived from adipose tissue), we believe that it is not a cell-specific event. Further optimization studies with different transfection agents, ratios of modified nucleosides, and mRNA/transfection reagent ratios are needed to understand this.
      To confirm the functionality of mRNA transfection, we chose an endogenously important RPE transcription factor: MITF. This gene is expressed from several promoters in an overlapping, cell-specific manner, resulting in different isoforms of MITF. It has been shown that, during the development of the murine RPE, the N-terminal 1B1b domain of MITF is crucial (
      • Bharti K.
      • Liu W.
      • Csermely T.
      • Bertuzzi S.
      • Arnheiter H.
      Alternative promoter use in eye development: the complex role and regulation of the transcription factor MITF.
      ). All MITF isoforms, except for MITF-M, contain this domain. In addition, it has been generally assumed that the M isoform is only expressed in melanocytes. However, recent reports have shown relatively high levels of the M-isoform in adult and prenatal human RPE cells as well as hESC-derived RPE cells (
      • Maruotti J.
      • Thein T.
      • Zack D.J.
      • Esumi N.
      MITF-M, a “melanocyte-specific” isoform, is expressed in the adult retinal pigment epithelium.
      ,
      • Capowski E.E.
      • Simonett J.M.
      • Clark E.M.
      • Wright L.S.
      • Howden S.E.
      • Wallace K.A.
      • Petelinsek A.M.
      • Pinilla I.
      • Phillips M.J.
      • Meyer J.S.
      • Schneider B.L.
      • Thomson J.A.
      • Gamm D.M.
      Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation.
      ). Whether different isoforms have a distinct or overlapping function in the adult RPE is not yet known. However, several reports have identified the 1B1b domain to be responsible for regulating cytoplasmic shuttling of MITF (
      • Lu S.Y.
      • Wan H.C.
      • Li M.
      • Lin Y.L.
      Subcellular localization of Mitf in monocytic cells.
      ,
      • Martina J.A.
      • Puertollano R.
      Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes.
      ). In accordance with this, our initial experiments with the transfection of MITF-A mRNA resulted in partial cytoplasmic localization of the MITF-A protein (data not shown). On the other hand, transfection of MITF-M mRNA gave rise to a strong nuclear localization of the protein, a necessary condition for correct evaluation of the transcription-regulating activity of MITF. Because of this, we chose the M isoform for comparison of the transcriptional functionality of the transfected mRNAs. Functional validation showed that both unmodified and modified mRNAs encoding MITF-M were able to activate many of the known MITF target genes, including TYR, TRPM1, and SILV. However, the MITF-M protein expressed from modified mRNA activated these genes more strongly than that expressed from unmodified mRNA. The reason for this is not clear, but because the expression levels of the encoded MITF-M protein were approximately equal, we speculate that the immune response generated by transfection with unmodified mRNA in some way interferes with the action of MITF, either directly or indirectly. It is known that exogenous dsRNA, as well as single-stranded RNA forming double-stranded secondary structures, acts as a ligand for Toll-like receptor 3 and, as such, may induce an innate immune response (
      • Alexopoulou L.
      • Holt A.C.
      • Medzhitov R.
      • Flavell R.A.
      Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3.
      ). TLR3 activation induces the recruitment of adapter molecules and kinases that lead to the translocation of the transcription factors IRF3 and NF-κB into the nucleus, where they promote the expression of TNFα and type I interferons such as IFNβ (
      • Alexopoulou L.
      • Holt A.C.
      • Medzhitov R.
      • Flavell R.A.
      Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3.
      ,
      • Vercammen E.
      • Staal J.
      • Beyaert R.
      Sensing of viral infection and activation of innate immunity by toll-like receptor 3.
      ). Considering that TLR3 is known to be expressed in the RPE (
      • Kumar M.V.
      • Nagineni C.N.
      • Chin M.S.
      • Hooks J.J.
      • Detrick B.
      Innate immunity in the retina: Toll-like receptor (TLR) signaling in human retinal pigment epithelial cells.
      ), our results are consistent with the current literature. The nuclear localization of RelA/p65 (an NF-κβ subunit) and IRF3, as well as the increased expression of IFNβ and TNFα in RPE cells transfected with unmodified mRNA may be attributed to TLR3 recognition of the transfected mRNA. Moreover, it has been reported that mRNA incorporating pseudouridine and/or methylated nucleosides is not efficiently recognized by TLRs (
      • Karikó K.
      • Buckstein M.
      • Ni H.
      • Weissman D.
      Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.
      ), which explains why our transfections with modified mRNA did not greatly induce the innate immune response.
      In conclusion, we have found that synthetic mRNA can be delivered effectively in hESC-RPE cells and that the expressed proteins are functional. Moreover, we have shown that synthetic modified mRNA can both bypass the innate immune response, therefore minimizing cytotoxic effects, and also generate a more biologically active protein, as shown when modified mRNA encoding MITF was transfected in hESC-RPE cells. Although the expression from the transfected mRNA is transient and, as such, might not be applicable at the present time for gene-based therapy treatments directly in patients, our study establishes a unique platform to use hESC-RPE cells and modified mRNA transfection for basic research, disease modeling, or as an approach to further develop therapeutic strategies for retinal degenerative disease treatments, such as cellular mRNA engineering, to improve successful cell transplantation.

      Acknowledgments

      We thank C. Tarantino, M. Díaz, L. Miquel, and the platforms at the Center of Regenerative Medicine in Barcelona for technical assistance.

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