Pelin ÜNAL, Mehmet YILDIZ, Ersin AKINCI, Gamze BADAKUL

In vitro transcription and validation of human pancreatic transcription factors' mRNAs

Direct reprogramming of pancreatic beta cells is of great interest due to its possible use in diabetes treatment. Various studies have demonstrated direct reprogramming of insulin expressing beta cells from other somatic cells by overexpressing important pancreatic transcription factors through viral vectors. However, concerns about using viral vectors in the clinic have prompted scientists to find new approaches for gene delivery and expression. In vitro synthesis and transfection of mRNAs is a safer strategy for ectopic gene expression. In this study we aimed to design and in vitro synthesize human pancreatic transcription factors mRNAs: Pdx1, Ngn3, and MafA. We also aimed to assess their expression efficiency in cultured cells. First we ligated the open reading frames of these genes to modified 5′ and 3′ UTRs. Ligation products were then cloned into a plasmid and subjected to in vitro transcription to get their corresponding mRNAs. Modified mRNAs were subsequently examined for their quality, quantity, and protein translation efficiency. In vitro-transcribed mRNAs were successfully transfected to the human fibroblast cells. Efficiently translated proteins were correctly localized into the nucleus. It is concluded that pancreatic mRNAs designed and in vitro-transcribed in this study can be used in further direct beta cell reprogramming studies.

PDF

___

Akinci E, Banga A, Greder LV, Dutton JR, Slack JM (2012). Reprogramming of pancreatic exocrine cells towards a beta (beta) cell character using Pdx1, Ngn3 and MafA. Biochemical Journal 442: 539-550.
Angel M, Yanik MF (2010). Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS One 5: e11756.
Arnold A, Naaldijk YM, Fabian C, Wirth H, Binder H, Nikkhah G, Armstrong L, Stolzing A (2012). Reprogramming of human Huntington fibroblasts using mRNA. ISRN Cell Biology 2012: 12.
Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003). Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17: 126-140.
Banga A, Akinci E, Greder LV, Dutton JR, Slack JM (2012). In vivo reprogramming of Sox9+ cells in the liver to insulin-secreting ducts. Proc Natl Acad Sci U S A 109: 15336-15341.
Boczkowski D, Lee J, Pruitt S, Nair S (2009). Dendritic cells engineered to secrete anti-GITR antibodies are effective adjuvants to dendritic cell-based immunotherapy. Cancer Gene Ther 16: 900-911.
Caiazzo M, DellAnno MT, Dvoretskova E, Lazarevic D, Taverna S, Leo D, Sotnikova TD, Menegon A, Roncaglia P, Colciago G et al (2011). Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476: 224-227.
Cao N, Huang Y, Zheng J, Spencer CI, Zhang Y, Fu JD, Nie B, Xie M, Zhang M, Wang H et al (2016). Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science 352: 1216-1220.
Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C (2004). Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303: 1529-1531.
Drews K, Tavernier G, Demeester J, Lehrach H, De Smedt SC, Rejman J, Adjaye J (2012). The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts. Biomaterials 33: 4059-4068.
Fischer A, Hacein-Bey-Abina S, Cavazzana-Calvo M (2011). Gene therapy for primary adaptive immune deficiencies. J Allergy Clin Immunol 127: 1356-1359.
Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, Türeci O, Sahin U (2006). Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108: 4009-4017.
Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M et al (2006). 5-Triphosphate RNA is the ligand for RIG-I. Science 314: 994-997.
Hu W, Qiu B, Guan W, Wang Q, Wang M, Li W, Gao L, Shen L, Huang Y, Xie G (2015). Direct conversion of normal and Alzheimers disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell 17: 204-212.
Jaenisch R, Young R (2008). Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132: 567-582.
Jiang J, Chan YS, Loh YH, Cai J, Tong GQ, Lim CA, Robson P, Zhong S, Ng HH (2008). A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10: 353-360.
Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008). Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16: 1833-1840.
Kawai T, Akira S (2007). Antiviral signaling through pattern recognition receptors. J Biochem 141: 137-145.
Knoepfler PS, Zhang XY, Cheng PF, Gafken PR, McMahon SB, Eisenman RN (2006). Myc influences global chromatin structure. EMBO J 25: 2723-2734.
Kondo T, El Khattabi I, Nishimura W, Laybutt DR, Geraldes P, Shah S, King G, Bonner-Weir S, Weir G, Sharma A (2009). p38 MAPK is a major regulator of MafA protein stability under oxidative stress. Mol Endocrinol 23: 1281-1290.
Li L, Allen C, Shivakumar R, Peshwa MV (2013). Large volume flow electroporation of mRNA: clinical scale process. Methods Mol Biol 969: 127-138.
Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA (2007). Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9: 625-635.
McConnell BB, Ghaleb AM, Nandan MO, Yang VW (2007). The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays 29: 549-57.
Miyagi S, Saito T, Mizutani K, Masuyama N, Gotoh Y, Iwama A, Nakauchi H, Masui S, Niwa H, Nishimoto M et al (2004). The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells. Mol Cell Biol 24: 4207-4220.
Pesce M, Scholer HR (2001). Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19: 271-278.
Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, Reis e Sousa C (2006). RIG-I-mediated antiviral responses to single-stranded RNA bearing 5-phosphates. Science 314: 997- 1001.
Plews JR, Li J, Jones M, Moore HD, Mason C, Andrews PW, Na J (2010). Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach. PLoS One 5: e14397.
Segre JA, Bauer C, Fuchs E (1999). Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22: 356-360.
Semsei I, Ma SY, Cutler RG (1989). Tissue and age specific expression of the myc proto-oncogene family throughout the life span of the C57BL/6J mouse strain. Oncogene 4: 465-471.
Su Z, Niu W, Liu ML, Zou Y, Zhang CL (2014). In vivo conversion of astrocytes to neurons in the injured adult spinal cord. Nat Commun 5: 3338.
Takahashi K, Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676.
Tavernier G, Wolfrum K, Demeester J, De Smedt SC, Adjaye J, Rejman J (2012). Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc. Biomaterials 33: 412-417.
Thacker EE, Timares L, Matthews QL (2009). Strategies to overcome host immunity to adenovirus vectors in vaccine development. Expert Rev Vaccines 8: 761-777.
Uematsu S, Akira S (2007). Toll-like receptors and Type I interferons. J Biol Chem 282: 15319-15323.
Van den Bosch GA, Van Gulck E, Ponsaerts P, Nijs G, Lenjou M, Apers L, Kint I, Heyndrickx L, Vanham G, Van Bockstaele DR (2006). Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells. J Immunother 29: 512-523.
Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal P, Smith ZD, Meissner A et al (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618-630.
Wilson A, Laurenti E, Oser G, Van der Wath RC, Blanco-Bose W, Jaworski M, Offner S, Dunant CF, Eshkind L, Bockamp E et al (2008). Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135: 1118-1129.
Yakubov E, Rechavi G, Rozenblatt S, Givol D (2010). Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 394: 189-193.
Yang Y, Akinci E, Dutton JR, Banga A, Slack JMW (2013). Stage specific reprogramming of mouse embryo liver cells to a beta cell-like phenotype. Mech Develop 130: 602-612.
Yisraeli JK, Melton DA (1989). Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol 180: 42-50.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R et al (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917-1920.
Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA (2008). In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455: 627-632.