title: Improved retroviral episome transfer of transcription factors enables sustained cell fate modification.
abstract: Retroviral vectors are versatile gene transfer vehicles widely used in basic research and gene therapy. Mutation of retroviral integrase converts these vectors into transient, integration-deficient gene delivery vehicles associated with a high degree of biosafety. We explored the option to use integration-deficient retroviral vectors to achieve transient ectopic expression of transcription factors, which is considered an important tool for induced cell fate conversion. Stepwise optimization of the retroviral episome transfer as exemplified for the transcription factor Oct4 enabled to improve both expression magnitude and endurance. Long terminal repeat-driven γ-retroviral vectors were identified as the most suitable vector architecture. Episomal expression was enhanced by epigenetic modifiers, and Oct4 activity was increased following fusion to a minimal transactivation motif of herpes simplex virus VP16. Based on kinetic analyses, we identified optimal time intervals for repeated vector administration and established prolonged expression windows of choice. Providing proof-of-concept, episomal transfer of Oct4 was potent to mediate conversion of human fibroblasts stably expressing Klf4, Sox2 and c-Myc into induced pluripotent stem cells, which were mainly free of residual Oct4 vector integration. This study provides evidence for suitability of retroviral episome transfer of transcription factors for cell fate conversion, allowing the generation of distinct patient- or disease-specific cell types.
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: J W
authors.initials: JW
authors.lastname: Schott
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Hoffmann
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Maetzig
authors.affiliation: Center for Integrated Psychiatry, Christian Albrechts University, Kiel School of Medicine, Kiel, Germany.
authors.forename: F-J
authors.initials: FJ
authors.lastname: Müller
authors.affiliation: 1] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany [2] Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Steinemann
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Zychlinski
authors.affiliation: 1] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany [2] Translational Hepatology and Stem Cell Biology, Hannover Medical School, Hannover, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Cantz
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany [3] Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Schambach
mesh_terms: D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D019426:Integrases / Q000235:genetics; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D014157:Transcription Factors; D019426:Integrases