Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA

Abstract

Induction of intrinsic liver regeneration is an unmet need that can be achieved by temporally activating key hepatocyte regenerative pathways. Here, we establish an efficient, safe, non-integrative method to transiently express hepatocyte-growth-factor (HGF) and epidermal-growth-factor (EGF) in hepatocytes via nucleoside-modified, lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) delivery in mice. We confirm specific hepatotropism of mRNA-LNP via intravenous injection of firefly luciferase encoding mRNA-LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes are transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA-LNP efficiently induce hepatocyte proliferation. In a chronic liver injury mouse model recapitulating non-alcoholic fatty liver disease, injections of both HGF and EGF mRNA-LNP sharply reverse steatosis and accelerate restoration of liver function. Likewise, HGF and EGF mRNA-LNP accelerate liver regeneration after acetaminophen-induced acute liver injury with rapid return to baseline ALT levels. This study introduces mRNA-LNP as a potentially translatable safe therapeutic intervention to harness liver regeneration via controlled expression of endogenous mitogens in vivo.

Fig. 1. A single IV injection of Luc mRNA-LNP induces robust and restricted luciferase activity in liver.

a Images of bioluminescence measurement from 5 h, day1 (d1) up to day 8 (d8) following tail vein (n = 2 mice) or retro-orbital (n = 6 mice) injection of Luc mRNA-LNP. Scales represent light intensity in photons/sec. b Quantification of light intensity (photons/sec) over time. Each dot represents one mouse. Source data are provided as a Source Data file.

Fig. 2. Identification of transfected liver cell types 5 h after IV eGFP mRNA-LNP injection.

a Co-immunostaining for eGFP (green) and liver cell type specific markers (red). ×200 magnification pictures and close-ups are shown. White arrowheads indicate co-stained cells. b Representative flow cytometry analyses showing percent of eGFP⁺ cells in CD11b⁺, CD45⁺, and CD31⁺ NPC fractions from mice injected with Poly(C) RNA-LNP or eGFP mRNA-LNP. All NPC fractions were gated for live cells present in Zombie NIR-negative populations. Data are presented as mean values ±  SEM for n = 3 mice per group. a Representative images from 3 mice injected with eGFP-mRNA-LNP and 2 mice injected with Poly(C) RNA-LNP. Source data are provided as a Source Data file. The scale bar represents 100 μm for all ×200 magnification images.

Fig. 3. A single IV injection of HGF mRNA-LNP induces significant hepatocyte proliferation in homeostasis.

a Immunostaining for HGF in mice sacrificed 5 h after IV injection of HGF mRNA-LNP or Poly(C) RNA-LNP. Images are ×100 magnification. b EdU staining on liver sections of mice 5, 24, or 48 h after injection with HGF mRNA-LNP or Poly(C) RNA-LNP. ×40 magnification pictures shown. Costaining for EdU/HNF4α (c) or EdU/CD45 (d) 48 h following injection of HGF mRNA-LNP or Poly(C) RNA-LNP. White arrowheads represent costained cells, while blue arrowheads represent single EdU stained cells. Close-ups from ×200 magnification pictures are shown. e Quantification of the absolute numbers of EdU⁺ cells at ×40 magnification from 2 mice per group per time point with 2–3 fields per mouse (n = 4–6) following injection of either Poly(C) RNA-LNP or HGF mRNA-LNP; the percentage of EdU⁺ cells that are HNF4α⁺ (hepatocytes) or CD45⁺ cells (immune cells) from 1 to 2 fields at ×100 magnification from 2 mice per group at 48 h post-injection (n = 3–4); and the calculated absolute numbers of EdU⁺HNF4α⁺ hepatocytes and EdU⁺CD45⁺ immune cells per ×40 magnification field at 48 h based upon the counted percentages (n = 4–6). Data are presented as mean values ± SEM. a–d Representative images from 2 mice injected with either Poly(C) mRNA-LNP or HGF mRNA-LNP per time point. P values were calculated by two-sided student’s t test, ***<0.0001, **<0.001, *<0.05, n.s. = not significant. Source data are provided as a Source Data file. The scale bar is included for each panel.

Fig. 4. HGF/EGF mRNA-LNP accelerate restoration of liver function during recovery from CDE-induced chronic liver injury.

a Injury scheme timeline: mice were fed CDD choline deficient diet for 1 week followed by three weeks of CDD diet supplemented with 0.1% ethionine (CDE). The CDE diet was then replaced with choline-sufficient diet. Mice were injected with either controls Poly(C) RNA or Luc mRNA-LNP, or HGF(H) and EGF(E) single or combined (H/E) mRNA-LNP at the end of the CDE diet (T0), followed by another injection 4 days into recovery (T4). b Bright-field images at ×100 magnification of Oil Red O staining showing lipid accumulation in hepatocytes at T2 and T8 in mice injected with either Poly(C) RNA-LNP or H/E mRNA-LNP. Representative images from 3 to 4 mice per group are shown. The scale bar represents 200 μm for all ×100 magnification images. Analyses of total serum cholesterol levels (c) and ALT levels (d) from 3 to 4 mice per group in duplicate or triplicate (n = 8–12) per time point treated with controls Poly(C) RNA-LNP or Luc mRNA-LNP, or single HGF(H), single EGF(E) or the combination H/E mRNA-LNP indicate sharp reversion of steatosis and restoration of basic levels of ALT after H/E mRNA-LNP injections. Data are presented as mean values ± SEM. Dark grey: T0, light grey: control RNA, light blue: single mRNA, dark blue: combined mRNA. P values were calculated by two-sided student’s t test, ***<0.0001, **<0.001, *<0.05, n.s. = not significant. Source data are provided as a Source Data file.

Fig. 5. HGF/EGF mRNA-LNP ameliorate liver function during continuous CDE-induced chronic liver injury.

a Injury scheme timeline: mice were fed CDD choline deficient diet for 1 week followed by continuous CDD diet supplemented with 0.1% ethionine (CDE) for 4 weeks. Mice were injected with either Poly(C) RNA-LNP or HGF/EGF(H/E) mRNA-LNP after 3 weeks of CDE diet (T0), followed by another round of injections 4 days later (T4). Mice were analyzed at T2 and T8, 2 days and 8 days after the first mRNA-LNP administration. b Bright-field images at ×100 magnification of Oil Red O staining show decreased lipid content in H/E mRNA-LNP treated mice at T8 compared to T2 and compared to control Poly(C) RNA-LNP treated groups. Representative images from 4 mice per group per time point are shown. The scale bar represents 200 μm for all ×100 magnification images. Analyses of total serum cholesterol levels (c) and ALT levels (d) in 3–4 mice per group in duplicate or triplicate (n = 8–12) per time point indicate significant release of cholesterol into the serum and amelioration of serum ALT to baseline levels in H/E mRNA-LNP treated mice compared to those in Poly(C) RNA-LNP treated group. Data are presented as mean values ± SEM. Dark grey: T0, light grey: control RNA, blue: combined mRNA. P values were calculated by two-sided student’s t test, ***<0.0001, **<0.001, *<0.05, n.s. = not significant. Source data are provided as a Source Data file.

Fig. 6. HGF/EGF mRNA-LNP accelerate liver recovery in the APAP-induced acute liver injury model.

a Mice were exposed to the Whitten effect and an overnight fast prior to a single injection of APAP (550 mg/kg). A single dose of control Poly(C) RNA-LNP or HGF/EGF(H/E) mRNA-LNP was administered 24 h following APAP injection. b–e Mice were analyzed 32 h and 48 h after APAP injection for necrosis with ×100 magnification bright-field images of H&E staining (b), serum ALT levels (c, 3-4 mice per group in triplicate (n = 9–12) per time point, except Poly(C) treated mice at 32 h for which 2 mice were used (n = 6)), and observation of TUNEL⁺ necrotic and apoptotic cells in central vein liver tissue areas (d), that were quantified per ×40 magnification field (e, 3 mice per group per time point with 2 fields per mouse (n = 6)). b, d Representative images from 3 to 4 mice per group per time point. c, e Data are presented as mean values ± SEM. Grey: control RNA, blue: combined mRNA. P values were calculated by two-sided student’s t test, ***<0.0001, **<0.001, *<0.05, n.s. = not significant. Source data are provided as a Source Data file. Scale bars are represented for each panel.