Construction and functional evaluation of a polycistronic modified mRNA for human iPS cell generation

Abstract

Background & Aim During the last decade, induced pluripotent stem cells (iPSCs) had an enormous impact on the progress of cell biology and regenerative medicine. Several approaches are accomplished for iPSCs induction. Modified mRNAs (mmRNA) has shown a potential to derive safe and high efficient integration-free iPSCs. However, low stability of mRNAs resulted in daily transfection that is costly and time-consuming. To overcome this limitation, we have constructed a polycistronic mmRNA containing WPRE element as a potential tool for safe iPSCs induction. Methods, Results & Conclusion Methods We developed a 2A-mediated polycistronic plasmid containing a single expression cassette with open reading frames of four human pluripotency transcription factors (LIN28, NANOG, SOX2, and OCT4) along with the EGFP coding sequence and WPRE element. We cloned the polycistronic DNA fragment in an appropriate vector containing T7 promoter, untranslated regions and poly (A) tail for in vitro transcription. Transcripts are produced using T7 RNA polymerase, modified nucleotides and cap analog. Phosphatase treatment was employed to reduce the immune responses. Evaluating integrity and quantity, the transcript was run on a denaturing agarose gel. Finally, mmRNAs were transfected into HEK293T cells and EGFP expression was assessed by fluorescent microscopy and flow cytometry. Results Primary polycistronic plasmid was successfully constructed. Correct orientation of each cloned fragments in the vector was confirmed using PCR and sequence analysis. Subsequently, the total ORF subcloned into another plasmid designed for IVT reaction, and the accuracy of new construct was confirmed using restriction digestion. By in vitro transcription, the mmRNA transcript was produced efficiently with expected size. Transfection of synthesised mmRNA into HEK293T cells was carried out and the fluorescent signal was effectively detected using fluorescent microscope and flow cytometry. The results demonstrated that mmRNA was efficiently expressed in HEK293T cells. Moreover, the presence of WPRE resulted in significantly enhanced mmRNA stability and higher level of protein expression. Conclusions We developed a polycistronic mmRNA for producing safe iPSCs. The cap, modified nucleotides and poly (A) tail in the mRNA enhanced its stability and increased translation efficiency. Moreover, WPRE element in the expression cassette increased the mmRNA stability and the duration of protein expression.