FGF21 and APOA1 mRNA-based therapies for the treatment of experimental acute pancreatitis

Abstract

Background: Acute pancreatitis (AP) presents a significant clinical challenge with limited therapeutic options. The complex etiology and pathophysiology of AP emphasize the need for innovative treatments. This study explores mRNA-based therapies delivering fibroblast growth factor 21 (FGF21) and apolipoprotein A1 (APOA1), alone and in combination, for treating experimental AP.

Methods: Liver-targeted lipid nanoparticles (LNP)-mRNA formulations encoding FGF21, APOA1, and a chimeric APOA1-FGF21, were first tested for protein expression and bioavailability in vitro and in mice fed a high-fat diet. Efficacy studies were performed in the caerulein-induced AP (Cer-AP) model, and a new AP model combining ethanol feeding with ethanol binge plus palmitoleic acid administration, the EtOH/POA-AP model. A single dose of the APOA1, FGF21, and APOA1-FGF21 LNP-mRNAs formulations was administered in both models. Serum levels of pancreatic lipase (LIPC), amylase (AMYL), and aspartate aminotransferase (AST), along with pancreatic tissue analyses using two histopathological scores were performed to evaluate treatment effects.

Results: In vitro studies demonstrated the translation and secretion of APOA1, FGF21, and APOA1-FGF21 proteins encoded by the LNP-mRNAs. In vivo, LNP-mRNA administration increased serum levels of the respective proteins in metabolically impaired (i.e. high fat diet-fed) mice. In the Cer-AP model, serum markers of pancreatic injury were similarly reduced when mice were treated with APOA1, FGF21, and APOA1-FGF21 LNP-mRNA, and this effect was also observed in the histopathological analyses. The EtOH/POA-AP model was more aggressive than the Cer-AP model. FGF21 and APOA1-FGF21 LNP-mRNAs were protective according to LIPC and AMYL serum levels, while APOA1 LNP-mRNA had little effect. On the other hand, histological improvements were more evident in mice receiving APOA1 LNP-mRNA. In the EtOH/POA-AP model, FGF21 and APOA1-FGF21 LNP-mRNAs reduced serum AST levels, indicating hepatoprotective activity.

Discussion: This proof-of-concept study demonstrates the potential of mRNA-based therapies delivering FGF21 and APOA1 in experimental AP. While individual treatments effectively reduced pancreatic injury, the APOA1-FGF21 fusion molecule did not exhibit superior activity. Liver-targeted LNP-mRNA administration may offer a promising approach for treating AP, leveraging endogenous production pathways for therapeutic proteins. Further research is warranted to elucidate the mechanisms underlying their therapeutic efficacy and optimize treatment regimens for clinical translation.

Keywords: APOA1; Acute pancreatitis; Cytoprotection; FGF21; Pancreatic injury; mRNA therapy.

Fig. 1

FGF21, APOA1, and fused APOA1-FGF21 human mRNAs are translated and secreted in mammalian cells. Schematic representation of the different proteins encoded by the tested mRNAs (A). HEK293T cells were transfected with 2.5 µg FGF21, human APOA1, and human fused APOA1-FGF21 mRNAs, and 24 h later, FGF21 was detected by Western blotting in cell lysates (left blots) and APOA1 in cell culture supernatant (right blot) (B). Representative blots are shown. Ponceau staining of membranes is shown to denote equal loading of the gels. SP: signal peptide; GS10: GS linker with 10 amino acids

Fig. 2

Metabolically impaired and overweight mice (H/S mice) administered with a single dose of human FGF21, APOA1, APOA1-FGF21 LNP-mRNAs expressed and secreted the corresponding proteins. Evaluation of human APOA1 protein levels in serum from mice after a single administration of human APOA1 LNP-mRNA, and evaluation of human FGF21 levels in serum from mice after a single administration of FGF21 or APOA1-FGF21 LNP-mRNAs at the indicated time-points (A). mRNA levels of the indicated genes in the epididymal white adipose tissue from H/S-treated mice 24 h after LNP-mRNAs administration (B). N = 3 mice in the Lean_Tris-Suc and H/S_Tris-Suc groups, and n = 5 in H/S_APOA1, H/S_APOA1-FGF21, and H/S_FGF21 groups. A and B: mean and SEM. * p-value < 0.05; H/S: high fat-fed and streptozotocin-treated; LNP: lipid nanoparticles; SEM: standard error of the mean

Fig. 3

Serum markers of AP and liver tissue damage are reduced in the Cer-AP model upon APOA1, FGF21, and APOA1-FGF21 LNP-mRNA treatments. Illustration of the experimental setup of Cer-AP model (Created in https://BioRender.com) (A). Serum APO-A1 (B) and FGF-21 (C) levels at 6-, 12-, and 24 h post-induction in the Cer-AP model treated with the LNP-mRNAs. Serum ALT (D), AST (E), AMYL (F), and LIPC (G) in Cer-AP-induced mice treated with the LNP-mRNAs. B and C: pooled controls from mice not administered with each molecule; p-value * < 0.05, ** < 0.01, *** < 0.001. Cer-AP: caerulein-induced acute pancreatitis; LNP: lipid nanoparticles; ALT: alanine aminotransferase; AST: aspartate aminotransferase; AMYL: amylase; LIPC: lipase

Fig. 4

Serum markers of AP and liver tissue damage are reduced in the EtOH/POA-AP model upon APOA1, FGF21, and APOA1-FGF21 LNP-mRNA treatments. Illustration of the experimental setup of the EtOH/POA-AP model (Created in https://BioRender.com) (A). Serum APO-A1 (B) and FGF-21 (C) levels at 6-, 12-, and 24 h post-induction in the EtOH/POA-AP model treated with the LNP-mRNAs. Serum ALT (D), AST (E), AMYL (F), and LIPC (G) in the EtOH/POA-AP-induced mice treated with the LNP-mRNAs. C and D: pooled controls from mice not administered with each molecule; p-value * < 0.05, ** < 0.01, *** < 0.001. EtOH/POA-AP: Ethanol/palmitoleic acid-induced acute pancreatitis; LNP: lipid nanoparticles; ALT: alanine aminotransferase; AST: aspartate aminotransferase; AMYL: amylase; LIPC: lipase

Fig. 5

Histopathological analyses of pancreatic tissues in the Cer-AP model treated with LNP-mRNAs. Representative images of pancreatic tissues for each model and treatment (H&E, scale bar = 100 μm) (A). Histopathological analyses of pancreatic tissues with two pancreatitis scoring systems: Schmidt score (B) and a simplified score (C) in the Cer-AP model. p-value * < 0.05, ** < 0.01, *** < 0.001. Cer-AP: caerulein-induced acute pancreatitis; LNP: lipid nanoparticles; H&E: hematoxylin-eosin

Fig. 6

Histopathological analyses of pancreatic tissues in the EtOH/POA-AP models treated with LNP-mRNAs. Representative images of pancreatic tissues for each model and treatment (H&E, scale bar = 100 μm) (A). Histopathological analyses of pancreatic tissues with two pancreatitis scoring systems: Schmidt score (B) and a simplified score (C) in the EtOH/POA-AP model. p-value * < 0.05, ** < 0.01, *** < 0.001. EtOH/POA-AP: Ethanol/palmitoleic acid-induced acute pancreatitis. LNP: lipid nanoparticles; H&E: hematoxylin-eosin

Fig. 7

Illustration depicting how acute pancreatitis disrupts the normal function of pancreatic acinar cells, which produce digestive enzymes released into the duodenum. Due to several risk factors, acute pancreatitis results in inflammation, edema, and acinar cell necrosis, leading to enzyme leakage into the bloodstream. Two experimental mouse models of acute pancreatitis were treated with LNP-mRNAs encoding APOA1, FGF21, or their fusion chimera. The LNP-mRNAs are targeted to the liver, from where the corresponding proteins will be produced and released into circulation. Treatment reduced inflammation, edema, and necrosis, improved enzyme secretion, and demonstrated prolonged therapeutic effects, suggesting significant potential for these mRNA therapies in managing acute pancreatitis. Created in https://BioRender.com