Creating anti-senescence cell fate by engineering the chromatin landscape

Persistent stress can shift cell fate in somatic cells, in which cells gain a new functional identity through 3D chromatin rewiring. Emerging high-resolution 3D epigenomic studies have uncovered the critical role of chromatin loops for cell identity by orchestrating functional transcription units. However, the mechanisms by which cells overcome epigenetic barriers remain elusive. We reason that by implementing diverse chromatin modifications within a specific biological context, we can engineer somatic cells with chromatin conformational landscapes that do not exist in vivo to adopt optimal cell fates. Here we choose the senescent microenvironment: senescent cells communicate with neighbouring cells by secreting soluble factors and direct contact, amplifying and maintaining senescent neches. Timely management of this process by immune clearance in vivo is critical as the accumulation of senescent cells promotes tissue degeneration and age-associated disorders. We aim to generate cell populations that actively engage with this challenging milieu by neutralising the senescence-associated secretory factors or targeting the senescent cells themselves. The engineered cells will constitute a novel anti-ageing therapeutic avenue. To manipulate the fates of off-the-shelf diploid cells, we propose two complementary approaches: Aim 1. Genetic Approach: Utilising genome-editing techniques, we aim to selectively disrupt functional chromatin loops in diploid cells. We devised a computational approach to estimate which loop anchors (particularly CTCF binding sites) are most likely to contribute to functional transcriptional units through a comprehensive analysis of Hi-C datasets. By targeting the predicted loop anchors, we will systematically engineer cells with combinatorial disruptions in these key regulatory units. Aim 2. Epigenetic Approach: We are developing novel chromatin manipulating probes, by engineering our unique panel of Mintbodies (GFP-scFV) that recognise specific epigenetic marks. The panel of Mintbodies will be fused with chromatin modulatory enzymes. We will stably express the Mintbody-derived chromatin manipulating probes (MCMP) in the diploid cells, establishing novel chromatin states that adapt to the specific microenvironment. Aim 3: Functional validation of the novel chromatin-modified cells with anti-senescence features in vitro and in vivo. We will first employ innovative in vitro evolution screening techniques in the presence or absence of ‘senescent cell feeders’, leveraging the loop-Perturb-seq (Aim 1) and MCMP libraries (Aim 2) and the advanced single-cell multi-target ChIL-seq (scmtChIL-seq), which allows for simultaneous mapping of CTCF and Pol-II binding sites in each cell. Through screening within the senescent microenvironment, we will identify the single-cell resolution trajectories of the cells with selective advantages in the senescent environment. We seek to identify cell populations exhibiting desirable anti-senescence traits and retrieve the specific combinations of CRISPR targets and MCMPs for direct cell engineering for usage in vivo. To functionally validate our engineered cells in vivo (Aim 3), we will focus on mouse models of ageing and sarcopenia, an ageing-linked disorder characterised by diminished skeletal muscle mass and strength. We will introduce the modified cells in mice and test for improvement in ageing markers and muscle regeneration. We have assembled a team with diverse expertise in biology, technology and mathematics. By integrating these two approaches, we will develop a therapeutic strategy of creating cells with alternative regulatory circuits and subsequent functionality, and gain insights into how chromatin modulation contributes to cell identity. While our goal is engineering somatic cells into anti-senescence entities, this strategy is readily applicable in other biological contexts, e.g., cells with improved differentiation potential.