(T1348) ULTRASLICE™ MRNA INCORPORATING 5-METHOXYURIDINE EFFICIENTLY INSERTS TRANSGENE SEQUENCES IN IPSCS WITH HIGH VIABILITY AND MITIGATES DSRNA PRODUCTION
Associate Scientist Factor Bioscience Inc., United States
Abstract: mRNA-mediated gene-editing allows for transient expression of editing proteins, reducing risks of insertional mutagenesis. However, mRNA can contain double-stranded RNA (dsRNA) byproducts of in vitro transcription (iVT). Previous research on mutant T7 RNA polymerases or modified nucleotides (NTs) to reduce immunogenicity has not focused on gene-editing. Since gene-editing-mediated insertion of transgenic sequences relies on cellular mechanisms of homology-directed repair, dsRNA-induced immune responses may limit editing efficiency. To address this challenge, we synthesized UltraSlice™ gene-editing mRNA with mutant T7 RNA polymerases and modified NTs to explore immunogenicity and editing efficiency in induced pluripotent stem cells (iPSCs). GFP-encoding mRNA was synthesized to analyze protein expression, dsRNA, and immunogenicity. RT-PCR of innate immune markers and dsRNA dot blot showed that the standard T7/canonical (AUGC) NT sample produced the greatest amount of dsRNA and induced a significantly higher immune response (p < 0.01) compared to samples synthesized with a mutant T7 and/or 5-methoxyuridine (5moU). This effect was consistent in iPSCs and mesenchymal stem cells (MSCs). Moreover, the standard T7/canonical NT condition produced 10-fold greater dsRNA than samples made with a mutant T7 and canonical NTs. While all synthesis conditions resulted in similar percentages of GFP-expressing cells when electroporated, median fluorescence intensity doubled in MSCs and iPSCs electroporated with mRNA synthesized with a mutant T7 or 5moU. Similar results were observed with UltraSlice™ gene-editing mRNA. iPSCs were co-electroporated with UltraSlice™-encoding mRNA and a GFP template for targeted insertion into TRAC. Mutant T7- or 5moU-containing mRNA maintained efficient editing when compared to mRNA made using standard conditions and outperformed mRNA synthesized with both a mutant T7 and 5moU by 4-fold. The iPSCs electroporated with these mRNA also maintained high viability (>85%) 48 hours post-electroporation. These results suggest that incorporation of 5moU alone is sufficient to reduce the generation of immunogenic dsRNA byproducts during iVT and enable efficient targeted insertion of transgene sequences in cells with high viability.
