title: Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts.
abstract: BACKGROUND
  By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required.
  
  
  METHODS
  Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins.
  
  
  RESULTS
  Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies.
  
  
  CONCLUSIONS
  The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.
authors.affiliation: Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Rostock, Germany.
authors.forename: Frauke
authors.initials: F
authors.lastname: Hausburg
authors.forename: Silke
authors.initials: S
authors.lastname: Na
authors.forename: Natalia
authors.initials: N
authors.lastname: Voronina
authors.forename: Anna
authors.initials: A
authors.lastname: Skorska
authors.forename: Paula
authors.initials: P
authors.lastname: Müller
authors.forename: Gustav
authors.initials: G
authors.lastname: Steinhoff
authors.forename: Robert
authors.initials: R
authors.lastname: David
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D002522:Chlorocebus aethiops; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D011560:Pseudouridine / Q000737:chemistry / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D014162:Transfection; D014780:Viruses / Q000235:genetics / Q000378:metabolism
chemical_list: D017570:MyoD Protein; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D011560:Pseudouridine; D049452:Green Fluorescent Proteins; D003562:Cytidine; D004247:DNA; C016568:5-methylcytidine