Identification of novel targets for inhibition of cellular senescence in Idiopathic Pulmonary Fibrosis

Cellular senescence is a process of cell cycle arrest which occurs naturally, but is increased in many diseases including Idiopathic Pulmonary Fibrosis (IPF). IPF is a progressive fibrotic disease of the lung with a mean survival time of 2.5–5 years from diagnosis and significant unmet medical need. Senescent cells are characterised by a distinct senescence associated secretory phenotype (SASP) and release pro-fibrotic secretory factors that are believed to contribute to lung fibrosis. In IPF, fibroblasts play a key role in the pathogenesis of the disease, so the aim of this project was to identify new drug targets to inhibit cellular senescence in a primary model of lung fibroblast senescence. This target identification campaign utilised three phenotypic library platforms, modulating protein function through different mechanisms to identify novel targets. The three platforms include: a whole genome CRISPR knockout library, a library of 1500 secreted proteins and a library of 450 fully functionalised fragments (FFF). These libraries were optimised for use in a DNA damage induced senescence assay, which uses high throughput confocal microscopy to detect etoposide induced changes in cellular senescence. This assay measures nuclear p21 expression, cellular area and nuclear area as markers of cellular senescence. By applying the library of secreted proteins, we identified proteins that either induced or inhibited senescence. Selected hits were profiled for their effects on SASP mediators and fibrotic markers, which identified a protein of interest which has not previously been linked with senescence. Screening of the FFF library (FFFs are small molecule fragments designed to enable target deconvolution by mass spectrometry) identified a hit, which was deconvoluted to identify a list of proteins for further validation. Application of the whole genome arrayed CRISPR screen in primary fibroblasts required significant assay development. Although editing had been established by electroporation, it was not feasible at whole genome scale. Therefore, we optimised a lipid based Cas9 mRNA and synthetic gRNA transfection method that could be scaled and automated in a high throughput manner. This editing protocol was combined and optimised alongside the etoposide induced senescence assay to obtain a 7 day assay procedure. The whole genome CRISPR screen has been completed and achieved a hit rate of 1-2% and identified both known and novel regulators of cellular senescence. This project has enabled target identification and validation in primary fibroblasts through the application of multiple library platforms each representing unique target opportunities. To our knowledge, this is the first time an arrayed CRISPR screen has been performed in primary human lung fibroblasts and together, these screens will identify new targets for the treatment of IPF.