Interferon regulatory factors 1 and 4 in T cell mediated immune modulation

The transcription factor interferon regulatory factor (IRF) 1 is essential for T helper cell 1 differentiation. Hereby, the respective Irf1 knockout mouse displays a severe immune defect and has an Th2 directed immune status per se. This effect is measurable by increased production of interleukin-4, a Th2 associated cytokine, and increased levels of immunoglobulin E. By this, the mouse is incapable to cope with intracellular pathogens and eventually dies from infection (e.g. Leishmania major infection). Allergies are also mediated by a Th2 driven immune response, therefore the question raised, whether this knockout mouse would react stronger in case of an induced allergy. To address this topic, the murine OVA model for acute asthma was used. Interestingly and despite wide-ranging analyses, it could be shown in this thesis work that the IRF1 deficiency did not result in a more severe asthma pathology. The herein presented data urgently suggest to reconsider the long-standing paradigm of asthma as only being a Th2-diven disease. Newly dicovered Th cell subsets support this opinion. The Irf1 knockout mouse not only has a defect in Th cell differentiation, but also clearly shows the requirement of IRF1 in CD8 development. The numbers of CD8+ cells in the periphery is extremely reduced, mainly due to a thymic misregulation of MHC class I. In order to reveal cell-specific functions of IRF1, a conditionally targeted mouse for Irf1 was successfully generated in the framework of this thesis. This mouse will help to address a variety of questions regarding IRF1 function in a given setting. Beside IRF1, another factor of the IRF family, namely IRF4 is important for Th differentiation. IRF4 has functions in Th2, Th9 and Th17 cell development, and the IRF4 deficient mouse is completely resistant to murine experimental autoimmune encephalomyelitis, a model for the human disease multiple sclerosis, due to the total incapacity to generate Th17 cells producing IL-17. Surprising data are shown in this thesis, in which γδ T cells from Irf4 knockout mice are nevertheless totally capable of producing IL-17. The underlying mechanism was closer investigated, and although it was not possible to reveal the actual pathway (which is apparently different from the one triggering IL-17 production in Th17 cells), the possibilities were narrowed down by excluding many other apparently obvious pathways

A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA

The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications. Keywords: double-stranded RNA, in vitro transcription, messenger RNA, nucleoside-modified RNA, RNA purification, cellulose-based purification, RNA immunogenicit

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics