Design, Construction and Functional Evaluation of a Polycistronic Modified mRNA with the Aim of Human iPS Cell Induction

Background and Aim: During the last decade, induced pluripotentstem cells (iPSCs) technology had an enormous impact on the progressof stem cell biology and personalized regenerative medicine. Severalmethodologies are accomplished to generate iPSCs. Among them,synthetically-modified mRNAs (mmRNA) have shown great potentialto derive safe and highly efficient integration-free iPSCs. However, lowstability of mRNAs has raised the necessity of daily transfection, whichis time-consuming. To overcome this limitation, in the current studywe have designed and constructed a polycistronic mmRNA containingWPRE element that improves the stability of the produced mRNA.Methods: Due to the stoichiometric expression of the reprogrammingfactors we developed a 2A-mediated polycistronic plasmid containinga single expression cassette with several open reading frames (ORFs) ofhuman pluripotency transcription factors (LIN28, NANOG, SOX2 andOCT4) along with an EGFP reporter gene and WPRE element. We clonedthe polycistronic DNA fragment in an appropriate vector containing aT7 promoter, untranslated regions and poly (A) tail used as a templateDNA for the transcription. During in vitro transcription (IVT), mmRNAsare synthesized using T7 RNA polymerase, modified nucleotides andcap analogs. Phosphatase treatment was then carried out to reduce theimmune responses. To evaluate the length, integrity and quantity of thetranscript, an aliquot of transcription reaction was run on a denaturing agarose gel. Finally, synthesized polycistronic mmRNAs were transfectedinto HEK293T cells and EGFP expression was monitored at defined timepoints by fluorescent microscopy and flow cytometry in order to evaluatethe functionality and stability of the mmRNAs.Results: Primary polycistronic plasmid consisted of four humanreprogramming factors and EGFP coding sequences have beenconstructed in Royan Institute, Previously. In this study, we successfullyinserted the WPRE element into this vector. Correct orientations of clonedfragment in the vector were confirmed by PCR and sequencing analysis.Subsequently, the total ORF subcloned into another plasmid designedfor IVT reaction, and its accuracy was confirmed by restriction digestion.After transcription reaction, the efficient production of mRNA transcriptwith expected length was confirmed by agarose gel electrophoresis. Aftertransfection of mRNAs into HEK293T Cells, the green fluorescent signalwas assessed by fluorescent microscopy and flow cytometry. The resultsdemonstrated that synthesized polycistronic mmRNA were efficientlyexpressed in HEK293T cells and also the presence of WPRE in thedownstream of the transgenes resulted in significantly enhanced mmRNAstability, which in turn led to a higher level of protein expression.Conclusion: In conclusion, we have developed a stabilized polycistronicmmRNA with potential application for the generation of human iPS cells.The application of the 5’cap, modified nucleotides and 3’poly-A tail inthe structure of synthesized mRNA enhanced its stability and translationefficiency. On the other hand, phosphatase treatment, which wasperformed after in vitro transcription removed the 5’triphosphates at theend of the uncapped mRNAs to reduce the cellular immune responses,and applied WPRE element in the expression cassette increased themmRNA stability as well as the yield of protein expression.

کلیدواژه ها:

iPSCs ، Modified mRNA ، Polycistronic ، 2A Peptide ، Woodchuck Hepatitis Virus Posttranscriptional Regulatory (WPRE) element