Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins.
BACKGROUND
Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death.


RESULTS
RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-beta (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division.


CONCLUSIONS
Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.
Angel
Matthew
M
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Yanik
Mehmet Fatih
MF
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D007113:Immunity, Innate / Q000235:genetics*; D007150:Immunohistochemistry; D016899:Interferon-beta / Q000235:genetics; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D050795:STAT2 Transcription Factor / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D034741:RNA, Small Interfering; D055748:SOXB1 Transcription Factors; D050795:STAT2 Transcription Factor; C494085:Stat2 protein, mouse; D015534:Trans-Activators; C484738:UTF1 protein, human; D016899:Interferon-beta; D017346:Protein Serine-Threonine Kinases