UKRI

The immune system is indispensable for our defense against infections, and plays an essential role in homeostasis, where compartmentalization and specialization are key aspects of every immune cell. Microenvironmental cues (e.g. stromal cell-derived signals) are integrated by the cell networks forming the immune system to maintain immune tolerance or promote immunity against an antigen. Recent findings suggest that neurons also contribute to curb the immune response; they produce neuropeptides that act directly on immune cells such as dendritic cells and neutrophils. However, our understanding of neuroimmune interactions is superficial: we ignore whether specialized cell subsets are responsible for integrating neural cues, we lack precision when studying these connections, and it is unknown whether neuroimmune exchanges are relevant in different tissues and immune reactions, as with the case of lung immune responses related to allergy and chronic obstructive disorders. Asthma has been shown to present a neurological component not yet entirely linked to the immune system. To gain insight into neuroimmune modulation, SIGNAL aims to elucidate the presence and function of neuro-sentinel innate lymphoid cells (nsILC) in the lung: ILC dedicated to detect and translate neuro-derived signals. ILC are tissue resident immune cells which are pivotal coordinators of immune homeostasis, bridging the innate and the adaptive immune system. Using a multidisciplinary approach by combining the selectiveness of optogenetics and chemogenetics with the unbiased power of detection of single cell genomics, and the comprehensiveness of mouse in vivo models, I have setup myself to discover and define nsILC in the context of type-2 responses in the lung. My work will develop our understanding of neuroimmune interactions, and their influence in allergy and asthma, potentially discovering pathways which will provide the context for new approaches in treating this type of disorders.

Electron cryo-microscopy (cryo-EM) is the fastest growing technique to explore the structure of biological macromolecules. To limit radiation damage, images are recorded under low-dose conditions, which leads to high levels of experimental noise. To reduce the noise, one averages over many images, but this requires alignment and classification algorithms that are robust to the high levels of noise. When signal-to-noise ratios drop, cryo-EM 3D reconstruction algorithms become susceptible to overfitting, ultimately limiting their applicability. The algorithms can be improved by incorporating prior knowledge. The most widely used approaches in the field to date incorporate the prior knowledge that cryo-EM reconstructions are smooth in a Bayesian approach. However, in terms of information content, the smoothness prior reflects poorly compared to the vast amount of prior knowledge that structural biology has gathered in the past 50 years. I aim to develop a computational pipeline that can exploit much more of the existing knowledge about biological structures in the cryo-EM structure determination process. I will express this prior knowledge through convolutional neural networks that have been trained on many reconstructions, and use these networks in novel algorithms that optimise a regularised likelihood function. Similar approaches have excelled in image denoising and reconstruction in related areas. Preliminary results with simulated data suggest that significant improvements beyond the existing methods are possible, both in computational speed and in signal recovery capabilities. The proposed methods will enable faster computations with less user involvement, but most importantly, they will extend the applicability of cryo-EM structure determination to many more samples, alleviating the existing experimental requirements of particle size, ice thickness and sample purity.

What defines the functional specialization of a cell is its unique proteome. Proteins need to be translated and properly folded to ensure their biological function. Several protein quality control pathways have arisen during evolution, some of them specialized in monitoring protein folding in specific organelles . As a result of ageing, animals lose their ability to protect their proteome, leading to the accumulation of protein aggregates and the onset of age-related disorders. The Unfolded Protein Response (UPR) of the Endoplasmic Reticulum (ER) is a central protein quality control mechanism that declines with ageing. This project will use the nematode Caenorhabditis elegans as a model to understand age-related proteostasis dysfunction. C. elegans offer many advantages over the use of vertebrate models, such as its short lifespan of about two weeks, reduced costs, and fewer ethical issues. We will focus on the UPR pathway, as changes in this pathway have already been implicated in the ageing process itself and in the onset of age-related disorders, such as Alzheimer’s disease. We propose to use CRISPR genome editing to create C. elegans transgenic reporter strains that enable us to track the activity of different UPR signalling molecules using microscopy and molecular biology techniques. We will monitor two central steps of the IRE-1/XBP-1 UPR pathway (1) the activation of the transmembrane sensor protein IRE-1 and (2) the biogenesis of the transcription factor XBP-1s. After age-related changes have been identified, we will use mutagenesis-based screening assays to screen for novel interventions that prevent UPR pathway shutdown. This research will contribute to a better understanding of why ageing occurs, as well as suggesting new therapeutic avenues for age-related disorders. This proposal will also be instrumental for the development and transference of new skills between the host and the applicant that will lead me towards independence.

Deciphering how nucleic acids replicated in the absence of genetically encoded enzymes is of critical importance to understanding the onset of Darwinian evolution. While much effort has been put into developing chemically-driven copying of RNA exploiting activated monomers, many unsolved issues stand in the way of achieving repeated cycles of non-enzymatic RNA replication. Non-enzymatic copying of a template strand results in the formation of an RNA duplex, which must then be denatured in order for subsequent rounds of replication to take place. Although RNA strands can be separated by heating, re-annealing kinetically outcompetes slow non-enzymatic copying, thus inhibiting RNA amplification. One unexplored solution to this problem is to physically separate melted strands of RNA so that re-annealing is not possible. Since all known living systems exploit lipid membranes, we propose to investigate whether protocellular compartments can facilitate the emergence of simplistic chemical systems that amplify RNA. Specifically, high temperatures are known to induce both RNA strand separation and bilayer defects, ultimately allowing for the partial leakage of RNA. If the transition temperature of the lipid membrane is higher than the melting temperature of the RNA, then subsequent slow cooling would recover the original impermeability of the membrane and give rise to a fraction of protocellular structures containing stochastic numbers of single RNA strands. At this stage, feeding with permeable activated short (oligo)nucleotides would lead to renewed copying of RNA. This highly original and multidisciplinary project combines the strength of organic and supramolecular chemistry to optimise prebiotic compartments with the power of in situ non-enzymatic RNA biochemistry to yield a project of excellent, innovative science that will exploit my expertise in protocellular systems while providing me extensive training in organic synthesis, chemical biology and biophysics.

The assembly of functional synthetic mammalian genomes is the most ambitious biotechnological challenge of the current century. Synthetic genomics has generated remarkable results in viruses and bacteria, but the de novo assembly of eukaryotic genomes is still limited to a few small chromosomes. Indeed, efficient delivery and genome engineering strategies for systematic genome replacement and assembly in higher eukaryotic cells are still missing. With my project RE-GENESis, I intend to make an essential contribution toward a more comprehensive understanding on the mammalian genome through the synthetic replacement of recoded loci at megabase-scale. My main goal is to demonstrate the feasibility of synthetic genomics and genetic code reprogramming at the megabase-scale in mammalian cells, using mouse embryonic stem cells to identify efficient delivery vectors, genome editing strategies and recoding schemes. I will use CRISPR-Cas in combination with large vectors to find new genome surgery strategies for genome recoding and gene correction and exploit them to replace the 2.4 Mb DMD (Duchenne Muscular Dystrophy)/dystrophin gene as a landmark application. The developed delivery and DNA replacement strategies will be also readily available for genome editing therapeutic approaches in order to correct most of the DMD disease-related mutations in human stem cells. RE-GENESis will thus pave the way for the effective deployment of synthetic genomics in high eukaryotes, expanding our understanding of complex eukaryotic genomes, delivering new tools to modify and regulate mammalian DNA and providing innovative solutions to treat genetic diseases, epidemics and ageing. This highly original and multidisciplinary project combines the strength of CRISPR-Cas with the power of large delivery vectors to yield a project of excellent, innovative science that will exploit my expertise in genome editing while providing me extensive training in genome recoding and synthetic genomics.