Messenger RNA (mRNA)‑based therapeutics confer less risk due to the transient nature of expression and with the success of the Covid‑19 vaccinations a greater confidence in their safety has resulted in more therapies progressing to the clinic. Liver fibrosis and cirrhosis remain an unmet need for treatment as cases are still increasing resulting in worldwide healthcare burdens. Until recently mRNA‑based therapies have not been evaluated in liver fibrosis/cirrhosis.
In this study, the authors examined the therapeutic utility of mRNA delivery in liver fibrosis and cirrhosis and demonstrated the efficacy of the human transcription factor hepatocyte nuclear factor alpha (HNF4A) encoding mRNA in the chronically injured liver:
Analysis of samples from human patients with fibrosis demonstrated a stage-dependent reduction of HNF4A expression in multiple cohorts. HNF4A protein levels in primary human hepatocytes from fibrotic patients was also reduced.
Four mouse models of liver injury, CCl4‑fibrosis, DDC, Mdr2-/- and CCl4‑cirrhosis, were utilised to investigate the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis.
HNF4-injected mice had reduced transaminases, bilirubin, and significantly reduced levels of hepatic collagen. qPCR analysis showed decreased expression of the fibrogenic marker genes, Col1a2, Col2a1 and Acta2.
Immunostaining revealed that, in both wild type and CCl4‑fibrotic mice, HNF4A was mainly expressed in hepatocytes rather than other hepatic cell types.
In vitro mRNA transfection experiments showed that restoration of HNF4A in fibrotic primary mouse hepatocytes rescued some impaired functions. This data was confirmed with repeated delivery of HNF4A mRNA lipid particles in CCl4- and DDC‑fibrotic mice.
Lipid particles of encapsulated HNF4A mRNA were internalised via endocytosis by hepatocytes and efficiently targeted murine fibrotic livers.
Microarray-based gene expression profiling, single cell RNA sequencing, and chromatin immunoprecipitation suggested that Paraoxonase 1 (PON1) is a direct target of HNF4A and contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of macrophages and hepatic stellate cells (HSC).
Gene expression analyses of IL6, iNOS, Arg1 and Fizz1 confirmed that HNF4A drives macrophages into the anti-inflammatory M2 phenotype. Likewise, exposure of HSC to HNF4A reduced fibrogenic markers such as desmin, αSMA and LOX.
Dose and schedule optimisation experiments in mice increased effectiveness of HNF4A mRNA delivery on liver fibrosis attenuation.
Serum cytokines demonstrated that either single or repeated injections of 2mg/kg of encapsulated mRNA was well tolerated and did not elicit substantial immune response in CCl4‑fibrotic mice.
Liver fibrosis and cirrhosis are current unmet medical needs. In this paper, the authors showed that restoration of a key gene, HNF4A, decreases fibrosis in multiple pre-clinical models. Therefore, this study provides a proof-of-concept that this new class of mRNA‑based therapy would be a promising strategy for reversing liver fibrosis and cirrhosis.
Yang T, Poenisch M, Khanal R, Hu Q, Dai Z, Li R, Song G, Yuan Q, Yao Q, Shen X, Taubert R, Engel B, Jaeckel E, Vogel A, Falk CS, Schambach A, Gerovska D, Araúzo-Bravo M, Vondran FWR, Cantz T, Horscroft N, Balakrishnan A, Chevessier F, Ott M, Sharma AD. Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model. Journal of Hepatology (2021)