Liver fibrosis gene therapy: Targeted ablation of portal fibroblasts – LIVES

Fibrotic diseases, notably liver diseases, are a major burden worldwide. Ablating myofibroblasts (MFs), the key effectors of fibrosis, has recently emerged as an innovative anti-fibrotic strategy. In liver fibrosis, MFs largely derive from hepatic stellate cells (HSCs) but also from portal fibroblasts. Partners 1&2, who developed scRNAseq analysis of liver mesenchymal cells in collaboration, uncovered portal fibroblasts with mesenchymal stem cell features (PMSCs) as a reservoir of highly proliferative and proangiogenic MFs (PMSC-MFs) (Lei et al., Hepatology 2022, DOI: 10.1002/hep.32456). Our postulate is that PMSC-MFs drive fibrosis progression and that it would be advantageous for the treatment of advanced fibrosis, to ablate this population rather than HSC-derived myofibroblasts (HSC-MFs), which promote liver regeneration. The aim of this proposal is to set up a preclinical protocol for anti-fibrotic gene therapy via targeted ablation of PMSC-MFs (or all-liver-MFs for comparison) in liver fibrosis. Relying on our previous RNAseq data, Asfalia Biologics, a start-up developing secured gene therapy based on temporal control of transgene expression (Partner 3), has undertaken the production of tools to target the conditional suicide gene iCaspase9 (iCasp9), to PMSCs/PMSC-MFs or to all-liver-MFs including HSC-MFs. Using scRNAseq and microRNAseq, we will further define the transcriptome demarcating PMSCs/PMSC-MFs or all-liver-MFs as compared to other liver cell types in mouse liver fibrosis in vivo. The promoter sequences of selected genes will be defined by chromatin accessibility using ATAC-seq and then synthetic promoters of reduced size but providing restricted expression of the transgene, will be cloned in lentiviral vectors to ensure cell-type-specific expression in transduced cultured cells including PMSC-MFs, HSC-MFs, Hepatocytes. Finally, the suicide gene iCasp9 and the reporter gene iRFP will be targeted to PMSC-MFs or to all-liver-MFs in the fibrotic liver of mice using an AAV6 vector. The expression of the transgenes will be restricted to PMSC-MFs or all-liver-MFs through vector tropism, transcriptional control and post-transcriptional silencing. The outcome will be evaluated on fibrosis and other aspects of liver wound healing including angiogenesis and liver regeneration. Targeting a new anti-fibrotic gene therapy to a MF subpopulation, critical in fibrosis progression, is a major technological and biomedical perspective.