Background & Aims

Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis.

Methods

We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation.

Results

Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells.

Conclusion

Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver.

Lay summary

Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

中文翻译：

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治疗性 HNF4A mRNA 在临床前模型中减轻肝纤维化

背景与目标

需要通过 mRNA 递送瞬时恢复蛋白质的损伤的治疗性靶向是一种有吸引力的方法，直到最近，该方法仍未得到很好的探索。在这项研究中，我们检查了 mRNA 递送对肝纤维化和肝硬化的治疗效用。具体而言，我们旨在证明人肝细胞核因子 α ( HNF4A ) mRNA 在纤维化和肝硬化小鼠模型中的治疗效果。

方法

我们在体外研究了通过HNF4A mRNA 转染对肝细胞功能的恢复，并通过递送包裹HNF4A mRNA的肝细胞靶向可生物降解脂质纳米颗粒 (LNP) 分析了多个小鼠模型中肝纤维化和肝硬化的衰减。为了确定潜在的作用机制，我们进行了基于微阵列的基因表达谱、单细胞 RNA 测序和染色质免疫沉淀。我们使用原代肝细胞和人类肝芽进行额外的功能验证。

结果

HNF4A mRNA 的表达导致体外纤维化原代鼠和人肝细胞的代谢活性的恢复。在 4 个独立的肝毒素和胆汁淤积诱导的肝纤维化小鼠模型中，LNP 包封的HNF4A mRNA 的重复体内递送诱导了对纤维化的强烈抑制。从机制上讲，我们发现对氧磷酶 1 是 HNF4A 的直接靶标，它通过调节肝巨噬细胞和肝星状细胞促进 HNF4A 介导的肝纤维化减弱。

结论

总的来说，我们的研究结果提供了HNF4A mRNA 疗法可用于治疗肝脏纤维化的第一个直接临床前证据。

总结

肝纤维化和肝硬化的医疗需求仍未得到满足，并导致全世界的高死亡率。在此，我们利用一种有前途的治疗方法来治疗肝纤维化和肝硬化。我们证明，通过包裹在脂质纳米颗粒中的 mRNA 恢复关键基因 HNF4A 可减少多种纤维化和肝硬化小鼠模型的损伤。我们的研究提供了概念验证，即 mRNA 疗法是逆转肝纤维化和肝硬化的一种有前途的策略。