The recently re-discovered interest in N ⁶ -methyl adenosine (m6A) - one of more than a hundred modifications found on eukaryotic mRNA (known as epi-transcriptomic codes) - is currently one of the most topical areas in science. The m6A methylation impacts on all aspects of cellular RNA metabolism and several physiological processes. Although less abundant than the m6A epitranscriptomic mark, the m1A methylation has recently also attracted interest due to its dynamic nature in response to physiological changes. We investigated the role of the YTH domain-containing m6A reader protein family and the m1A eraser ALKBH3 on the expression of a transgene in mammalian cells. We present the first evidence that expression of a transgene is subjected to co-ordinated regulation by both m6A and m1A regulators. In addition, we provide genetic data implicating that the m6A reader YTHDF2 can read m1A site. Furthermore, we show that the m1A eraser ALKBH3 is a target of m1A methylation.

Lentiviral transduction remains the gold standard in adoptive modified cellular therapy, such as CAR-T; however, genome integration is not always desirable, such as when treating non-fatal autoimmune disease or for additional editing steps using CRISPR to produce allogeneic CAR-modified cells. Delivering in vitro-transcribed (IVT) mRNA represents an alternative solution but the labile nature of mRNA has led to efforts to improve half-life and translation efficiencies using a range of approaches including chemical and structural modifications. In this study, we explore the role of N⁶–methyladenosine (m6A) in a CD19-CAR sequence when delivered to T cells as an IVT mRNA. In silico analysis predicted the presence of four m6A consensus (DRACH) motifs in the CAR coding sequence and treating T cells with an inhibitor of the m6A methyltransferase (METTL3) resulted in a significant reduction in CAR protein expression. RNA analysis confirmed m6A bases at three of the predicted sites, indicating that the modification occurs independently of nuclear transcription. Synonymous mutation of the DRACH sites reduced the levels of CAR protein from 15 to >50% depending on the T cell donor. We also tested a panel of CAR transcripts with different UTRs, some containing m6A consensus motifs, and identified those which further improved protein expression. Furthermore, we found that the methylation of consensus m6A sites seems to be somewhat sequence-context-dependent. These findings demonstrate the importance of the m6A modification in stabilising and enhancing expression from IVT-derived mRNA and that this occurs within the cell, meaning targeted in vitro chemical modification during mRNA manufacturing may not be necessary.

Recent bulk analysis of Chinese hamster ovary (CHO) cell mitochondrial DNA revealed widespread heteroplasmy across cell lines and even within clones of the same parental host. To address this, we applied our previously developed single‐cell mtDNA sequencing (scmtDNAseq) method to 84 single CHO cells. We identified widespread intercellular heteroplasmy across the CHO cell population and predicted possible phenotypic impacts. 3/11 (27%) of the most variable mutations were only identified by scmtDNAseq, indicating greater resolution when compared to bulk cell analysis. Single‐cell RNAseq (scRNAseq) was also performed at the same time point and, compared to scmtDNAseq, significant differences in intercellular heteroplasmy were observed. Using an inducible mAb expression system demonstrated that short‐term additional biosynthetic burden of exogenous protein production had little impact on intercellular heteroplasmy. We additionally monitored bulk heteroplasmy over 38 days, reflecting the typical timespan from vial thaw to production vessel in a Biopharmaceutical upstream cell culture process. We observed minimal change in heteroplasmy, finding no evidence that a mAb‐producing CHO cell line develops impactful changes in heteroplasmy over that timeframe. This would suggest that for our cell line, the heteroplasmy profile established on Day 1 should be maintained throughout a full fed‐batch bioprocess run.