In vivo anti-FAP CAR T therapy reduces fibrosis and restores liver homeostasis in metabolic dysfunction-associated steatohepatitis
In this study, we aimed to determine the efficacy of in vivo chimeric antigen receptor (CAR) T cell therapy, generated by targeted lipid nanoparticles (t-LNPs), as an anti-fibrotic in metabolic dysfunction-associated steatotic liver disease. Hepatic fibrosis is a key predictor of mortality in liver disease, driven by fibrogenic hepatic stellate cells (HSCs). In heart, chimeric antigen receptor (CAR) T cells targeting fibroblast activation protein alpha (FAP) reduce murine cardiac fibrosis. However, the value of this approach in liver is unknown. We explored the anti-fibrotic potential of in vivo-generated anti-FAP CAR T cells in metabolic dysfunction-associated steatohepatitis (MASH), a highly prevalent disease with no approved anti-fibrotic therapies. We first established that FAP expression in both human and murine MASH is specific to HSCs. We then used flow cytometry, Sirius Red morphometry, digital pathology analysis, and single nuclear RNA-sequencing to assess the impact of anti-FAP CAR T cell therapy on murine MASH. Anti-CD5 targeted-LNPs carrying anti-FAPCAR mRNA generate activated, transient anti-FAP CAR T cells, which significantly reduced fibrosis by depleting pro-fibrogenic HSCs, and by modulating immune cells, endothelial cells and hepatocytes in a non-cell autonomous manner to mitigate inflammation and restore hepatic homeostasis. These findings reinforce the potential of in vivo CAR T therapy to attenuate a highly morbid and pervasive liver disease through its integrated, multicellular salutary effects. Overall design: Samples used were frozen minced murine liver (left lobe). There are four expeirmental groups: Chow diet + vehicle, FAT MASH model + vehicle, FAT MASH model + IgG-FAPCAR LNP, and FAT MASH model + CD5-FAPCAR LNP. About 10-15mg of snap frozen liver tissue from four representative mice per group were pooled. Samples were prepared via an established protocol for nuclei isolation from murine MASH tissue(36). Solutions used included 2xST buffer (292 mM NaCl (Thermo Fisher Scientific, cat. no. AM9759)), 20 mM Tris-HCl pH 7.5 (Thermo Fisher Scientific, cat. no.15567027), 2 mM CaCl2 (VWR International Ltd, cat. no. 97062-820) and 42 mM MgCl2 (Sigma Aldrich, cat. no. 1028) in DNA/RNAse free water. 0.03% TST was prepared by adding 5mL of 2xST, 83uL Ribolock,83uL 2%BSA, 300uL of 1% Tween20, 4.6mL of H2O. The samples used for snRNAseq Chromium 3' GEX were from snap frozen Chow + saline, MASH + saline, MASH + IgG-FAPCAR, and MASH + CD5-FAPCAR livers, about 3 months after euthanasia and cryopreservation. Each sample was chopped for 10 minutes over ice and passed through a strainer with 1mL TST. Samples were spun down at 500xg for 5 minutes at 4oC, supernatant was aspirated. Pellet was resuspended by pipetting 30 times with P1000 tips in 200uL of 0.4% BSA/PBS and then brought to the Genetics Core.