(474e) Cell-Free Production of Nona-Arginine Transcription Factor Fusion Proteins for Non-Viral Modulation of Gene Expression

Ectopic expression of a defined set of transcription factors (TFs) can reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, human iPSCs have been successfully generated using lentiviruses, retroviruses, transposons, and recombinant proteins. Nucleic acid-based methods raise concerns about mutagenesis, so it would be desirable to directly administer the TFs as proteins. The Kim group at Harvard recently showed that polyarginine peptide conjugation can deliver recombinant TF cargoes into cells and reprogram human fibroblasts into iPSCs. However, the protein-based approach requires a large amount of protein due to its low reprogramming efficiency. It is difficult to produce large amounts of fusion TFs using bacterial hosts due to product insolubility. Thus, we explored the use of cell-free protein synthesis (CFPS) as an alternative method for producing fusion TFs.

Using an E. coli-based CFPS system, we successfully expressed the set of six human transcription factors, OCT4, SOX2, C-MYC, KLF4, NANOG, and LIN28, as fusion proteins with a nona-arginine protein transduction domain. Then, we showed that R9-OCT4, R9-SOX2, and R9-NANOG retained their cognate DNA binding activities and R9-NANOG translocated across the plasma and nuclear membranes of fibroblasts. R9-SOX2 also modulated gene expression, exerting transcriptional activity on a known downstream target. In this talk, we will discuss the first step toward realizing a non-viral approach for generating iPSCs: developing and characterizing a platform for delivering transcription factors to the nucleus.