Regulatory workshop on challenge strain development and GMP manufacture - A stakeholder meeting report.

Within the Innovative Health Initiative (IHI) Inno4Vac CHIMICHURRI project, a regulatory workshop was organised on the development and manufacture of challenge agent strains for Controlled Human Infection Model (CHIM) studies. Developers are often uncertain about which GMP requirements or regulatory guidelines apply but should be guided by the 2022 technical white paper "Considerations on the Principles of Development and Manufacturing Qualities of Challenge Agents for Use in Human Infection Models" (published by hVIVO, Wellcome Trust, HIC-Vac consortium members). Where those recommendations cannot be met, regulators advise following the "Principles of GMP" until definitive guidelines are available.

Epigenetic reprogramming of airway macrophages promotes polarization and inflammation in muco-obstructive lung disease.

Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease (Scnn1b-transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization.

Queuosine-modified tRNAs confer nutritional control of protein translation.

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q-tRNA levels promote Dnmt2-mediated methylation of tRNA Asp and control translational speed of Q-decoded codons as well as at near-cognate codons.

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs.

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress and metabolic disorders. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications.

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs.

Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution.

Epigenetic regulation by heritable RNA.

Genomic concepts are based on the assumption that phenotypes arise from the expression of genetic variants. However, the presence of non-Mendelian inheritance patterns provides a direct challenge to this view and suggests an important role for alternative mechanisms of gene regulation and inheritance. Over the past few years, a highly complex and diverse network of noncoding RNAs has been discovered.

RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype.

RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.

The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation.