Inclusion of retinoic acid but not retinol improves mRNA delivery to fibrotic livers
We hypothesized that LNP-mediated mRNA expression in the liver would be reduced in models of chronic liver inflammation.17 To test this, we delivered a model mRNA encoding luciferase (mLuc) using clinically-approved LNPs composed of ALC-0315, SM-102 and MC3 in wild type (WT) animals and those with chronic liver inflammation (Fig. 2a-c). mLuc LNPs were administered via intravenous (i.v.) injection to an experimental late-stage liver fibrosis model induced by 6-week administration of tetrachloride (CCl4) and a NASH hamster model induced by 10-week choline deficient high fat diet (CDHFD) treatment (Fig. 2b, c, Fig. S1, S2). We observed a 6 ~ 20- fold decrease in mLuc expression delivered by all three types of LNPs in fibrotic models compared to WT controls.
Next, we designed a library of LNPs which would enhance mRNA delivery to hepatic stellate cells (HSCs) that are known to be abundant in fibrotic livers. Instead of employing a complicated post-fabrication surface modification strategy22, we selected to add an additional component to the LNP which could aid selective accumulation of LNPs in HSCs. HSCs are primarily responsible for retinoid storage.23 In vivo, retinol and selected retinol metabolites bind to the serum protein RBP-4 (Fig. 2d), which then facilitates cellular endocytosis in HSCs.24 We therefore developed LNPs containing retinoids and retinoid derivatives spanning four main sub-classes; (I) natural retinols, (II) natural retinol acids, (III) aromatic retinol acids, and (IV) retinol esters (Fig. 2d). These included first generation retinol derivatives which preserved the cyclohexane ring of natural vitamin A, such as retinol, fenretinide and 4-keto-retinol (with a hydroxyl end, group I); all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA) and 9-cis-retinoic acid (9-CRA) (with a carboxylic end, Group II), and acetyl retinol (with an ester bond, group IV). We also included a number of second generation of derivatives which had aromatic modifications in the cyclohexane ring area, including acitretin (A-VA) and bexarotene (carboxylic acid derivative, group III) and etretinate (ester derivatives, group IV). The second generation derivatives have showed selective binding to intracellular retinoid X receptors (RXRs) or retinoid acid receptors (RARs).25 However, the affect of these modifications on RBP-4 binding and uptake is not well understood. We measured the binding of these retinoid derivatives to RBP-4 (Table. S1). Most of the first-generation retinoids exhibited high binding affinity (Kd = 0.5 ~ 5µM), consistent with previous report,26 while the second generation showed moderate binding (Kd = 30 ~ 60 µM).
Based on these findings, we incorporated all retinoid derivatives into LNP formulations containing ALC-0315 as the ionizable cationic lipid. Retinoid derivatives could be directly incorporated into the lipid bilayer of LNPs due to the hydrophobic cyclohexane/aromatic ring and alkene chains (Fig. 2d).27 Retinol derivatives were included at 5 mol% to 25 mol% within the cholesterol component, other original lipid ratio was maintained (Table S2). We evaluated the transfection efficiency of mRNA encapsulated LNPs in aHSCs (using LX-2 cell line as model) and primary hepatocytes (both healthy and fatty hepatocytes). Results shown in Fig. 2e indicate that incorporating carboxylic acid retinol derivatives, as opposed to alcohol or ester derivatives, significantly enhanced mRNA delivery in aHSCs (Fig. S3). Notably, ATRA, 13-CRA and 9-CRA showed a dose-dependent increase in expression, with 5.4-, 4.1- and 4.2- fold increase of expression in aHSCs at 25 mol% incorporation, respectively. The acidic aromatic derivative A-VA and bexarotene showed 5.3- and 4.7- fold increase of mRNA delivery in aHSCs and plateaued at lower incorporation levels (~ 15 mol%). However, alcohol derivatives (retinol, fenretinide, and 4-keto-retinol) and ester derivatives showed comparable or decreased mRNA expression in aHSCs as compared to the original ALC-0315 formulation. Enhanced protein expression was not observed in healthy or fatty primary hepatocytes treated with retinoid derivative LNPs (RD-LNPs).
To validate these trends in vivo, RD-LNPs containing ATRA, bexarotene, A-VA and retinol (all at a 25 mol% replacement of cholesterol, Table S2) were formulated and compared to control ALC-0315 LNPs. The particle sizes of all five formulations were around 100 nm, with a polydispersity index (PDI) below 0.1. They exhibited encapsulation efficiency over 70% and a slightly negative charge (Fig. S4, Table S2). We injected these particles into mice that were pre-treated with CCl4 for 4 weeks. We observed that ATRA, A-VA and bexarotene LNPs significantly improved luciferase expression in fibrotic liver rather than the retinol LNPs, with ~ 8.0-, 3.7- and 2.8- fold increase in luciferase expression compared to the original ALC-0315 formulation in fibrotic livers, respectively (Fig. 2f, g, Fig. S5). This observation aligned with the in vitro transfection study conducted in LX-2 cells (Fig. 2e), highlighting the potential of targeting aHSCs for LNP delivery in fibrotic livers.
We found these trends could be generally applied to more aggressive fibrosis models and were also applicable to other commercial cationic LNP formulations (MC3). In mice treated with CCl4 for 6 weeks, ATRA LNPs enhanced protein expression ~ 10- fold compared to the ALC-0315 formulations, however this increased expression was not observed in WT mice (Fig. 2h). Over 95% of mRNA was expressed in liver rather than other organs (Fig. 2i, Fig. S6). The same trend was also observed in an MC3 formulation containing ATRA, demonstrating the universal effectiveness of carboxylic RD-NPs, particularly ATRA, in facilitating LNP delivery to fibrotic livers (Fig. S7).
Finally, we established a hamster CDHFD-induced NASH model as a more clinically relevant system. We then compared luciferase expression in these hamsters treated with ATRA-containing mLuc LNPs via jugular vein injection, or standard ALC-0315 mLuc formulations. Results were similar to CCl4 treated mice: we observed a 9.7- fold increase in expression in NASH hamster liver when ATRA was added to the original LNP formulations (Fig. 2j, Fig. S8). Together, these data demonstrate improved LNP delivery and mRNA translation to fibrotic liver in vitro and in vivo through incorporation of carboxylic retinoids in the LNP formulations.
Rearrangement of retinoic acid in LNP facilitates endocytosis and endosomal release of RNA in aHSCs
To validate the role of aHSCs in mediating enhanced mRNA expression in fibrotic livers treated with RD-LNPs, we looked protein expression in CCl4 treated tdTomato reporter mice (Fig. 3a). Following the induction of fibrosis, we administered LNPs encoding for cre-recombinase mRNA (mCre LNPs). The tdtomato mice carry a LoxP flanked stop cassette mutation, and upon expression of Cre, the cells express tdTomato (Fig. 3a). We used flow cytometry to quantify the transfection efficiency of ALC-0315 LNPs and ATRA LNPs in different cells within the fibrotic livers. No significant difference was observed in the expression of tdTomato in the leucocyte population. However, approximately 40%, over 2- fold increase in the number of tdTomato+ cells were observed in HSC-like population treated with ATRA LNPs compared to those treated with ALC-0315 LNPs (Fig. 3b, Fig. S9). This result aligns with the in vitro study and suggests that ATRA LNPs tend to accumulate in the fibrotic area (Fig. 3c). In a separate study, we evaluated the co-localization of luciferase and alpha-smooth muscle actin (α-SMA, a marker for fibrosis), using immunofluorescence (IF) staining. Consistently, we observed that most of the expressed luciferase was present in α-SMA+ fibrotic area in mice treated with ATRA LNPs (Fig. 3d).
We next investigated the rationale of acidic retinoid derivatives in facilitating mRNA delivery to HSCs. BODIPY-labeled LNPs were used to assess cellular uptake through high-content microscopy (for LX-2 HSCs) and flow cytometry (for primary hepatocytes). The transfection time was limited to 1.5 h to avoid non-specific lipofection that is often observed with longer incubation time.28 To explore the potential mechanisms of endocytosis, the cells were treated with small-molecule inhibitors of clathrin/caveolae-mediated endocytosis and macropinocytosis prior to LNP treatment (Fig. 3e, Fig. S10). Specifically, ATRA and A-VA LNPs showed 1.5 ~ 2- fold higher uptake compared to ALC-0315 LNPs in aHSCs (LX-2 cells). Retinol, Acetyl-Retinol and Etretinate LNPs exhibited slightly lower but comparable uptake to ALC-0315 LNPs. In contrast, ATRA did not significantly facilitate LNP uptake in both WT and fatty hepatocytes (Fig. S12). Macropinocytosis was identified as a major pathway for LNP uptake for all LNPs tested, consistent with previous reports.29, 30 In a separate study, we also knocked down the RBP-4 receptor STRA6 with siRNA 24 h prior to adding LNPs (Fig. S11) in aHSCs. Interestingly, knockdown of STRA6 significantly reduced the uptake efficiency of retinoic acid LNPs (ATRA and A-VA LNPs) by approximately 2 ~ 3- fold in aHSCs. A light, non-significant decrease was observed in the retinol or retinol-ester groups (Acetyl Retinol and Etretinate LNPs). In contrast, STRA6 did not significantly affect hepatocyte uptake of ATRA LNPs (Fig. S12). This suggests that only the acidic retinoid derivative LNPs significantly rely on STRA6 for enhanced endocytosis in aHSCs. To further examine whether the interaction between RBP-4 and STRA6 facilitate the endocytosis of retinoic acid LNPs in LX-2 cells, we supplemented the cells with additional RBP-4 protein. This led to a slight but significant increase in the ATRA LNP-treated groups, further supporting the role of acidic retinoid in enhancing LNP binding to HSCs through the RBP-4 -STRA6 pathway (Fig. S10). We then used MST to measure the binding affinity between RBP-4 protein and mRNA loaded or empty LNPs. As expected, ALC-0315 LNP without added retinoids did not bind with RBP-4 protein (Fig. S13); however, the addition of retinoids improved LNP binding to RBP-4. Interestingly, encapsulating mRNA into retinol LNPs didn’t change the binding affinity, whereas adding of mRNA into ATRA LNPs led to approximately a 10- fold increase in the binding affinity with RBP-4 (Fig. 3f and g). This suggests lipid organization in ATRA LNPs is altered following the encapsulation of mRNA. We hypothesized that it could be due to charge mediated repulsion and lipid re-arrangement during LNP assembly. Briefly, the carboxylic acid derivative has a pKa around 4–5 (Table S1). During LNP synthesis and dialysis, the pH switches from 4 to 7. Consequently, the retinoic acidic derivative could be rearranged into the LNP surface to minimize negative charge interactions between the negatively charged mRNA and carboxylic acid derivative. This would make the carboxylic acid more accessible to RBP-4, impacting binding affinity and the associated endocytosis pathways (Fig. 3p). To gain further insights into the LNP structure, we employed Small-Angle Neutron Scattering (SANS) as previously described.16 The distribution of the ATRA within LNPs was elucidated by varying the content of deuterated water (D2O) to match the scattering length densities of different region of the particle (Table S9). Using a core-shell particle model to fit the SANS data, the results suggested that at least 70% of deuterium-labeled ATRA was preferentially located in the outer shell region, supporting our hypothesis (Fig. 3h)
Adding exogeneous RBP-4 to cell culture medium only slightly increased mRNA expression in cells treated with ATRA LNPs, indicating that other mechanisms may mediate the enhanced expression of mRNA. We next looked into the endosomal release kinetics of mRNA LNPs. We labeled LNPs with BODIPY-lipid and Cy3-mRNA, and tracked the intracellular transport of LNPs using confocal microscope via an Airyscan detector unit. We observed a significant dissociation of mRNA from dye-labeled LNP 2 h post-uptake in LX-2 cells (Fig. 3i), suggesting rapid dissembly and release of mRNA from endosomes in cells treated with ATRA LNP group. In contrast, around 80% of mRNA was still co-localized with LNPs in other groups. To understand the accelerated lipid-dissociation and mRNA release in more detail, we isolated the endosome compartment from fibroblasts and hepatocytes and performed lipidomic analysis (Fig. 3j, Fig. S14, S15). The result of lipidomics revealed that hepatocyte endosome has a lower saturation lipid content compared to HSCs’, suggesting that HSCs possess more rigid endosomal membranes. To mimic different properties on membrane packing, we created polymer-tethered lipid bilayer system using simplified lipid components, i.e., with 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) providing the net negative charge, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) constituting the main bilayer structures (Fig. 3k).31 The ratio of DOPC and DPPC was tuned to match the saturation levels of the endosomal membranes from HSCs and hepatocytes (Fig. 3l). Membrane rigidity was confirmed via measuring the diffusion coefficient using fluorescent correlation spectroscopy (Fig. S16). We used Texas-red PE labeled LNPs encapsulated mRNA to test the fusion kinetics between the artificial bilayer and LNPs. Interestingly, all three types of LNPs efficiently and rapidly fused within the soft membrane mimicking hepatocyte endosomes, with ATRA exhibiting slightly faster and more complete fusion (> 90% fused) (Fig. 3m, n, Video S1). However, in the rigid membrane mimicking HSC endosomes, almost no fusion events were observed for ALC-0315 LNPs (Fig. 3m, n, Video S2). Nevertheless, ATRA LNPs exhibited over 50% fusion at the end point (Fig. 3m), with faster diffusion compared to other LNP treated membranes (Fig. 3o). Additionally, the diffusion coefficient of the acceptor artificial membrane was significantly increased after treating with ATRA LNPs, suggesting lipid protrusion and mixing may play a rule (Fig. S16). Thus, we further proposed that the charge-mediated repulsion of ATRA to the outer shell of LNP might facilitate lipid protrusion and sprouting, ultimately enhancing endosomal escape (Fig. 3p). Overall, these findings enhance our understanding of the mechanisms underlying the improved transfection efficiency observed with retinoic acid LNPs targeting HSCs.
mRNA encoding collagen binding recombinant proteins improved retention in fibrotic region
Following the successful accumulation of mRNA LNPs in HSCs and the site-specific expression of protein in fibrotic liver regions, our next objective was to evaluate the strategy to retain the therapeutic protein in the fibrotic livers. The therapeutic peptide utilized in our study was a peptide hormone RLN, with anti-fibrotic effects that has been clinically tested for treating cardiovascular diseases. We aimed to anchor this protein in fibrotic lesions in the liver to enhance it’s local expression (Fig. 4a). Through RNA sequencing of WT hamsters and hamsters with NASH, we identified col1α1 and col1α2 as major ECM proteins significantly increased in NASH livers (Fig. S17).32 We decided to add ECM binding domains to the RLN hormone to evaluate if mRNA modifications could be used to enhance protein retention in the fibrotic livers. For initial screening, we chose 11 collagen binding domain (CBD) sequences derived from endogenous proteins such as decorin, fibronectin, osteopontin and others (Fig. 4b).33–36 Naturally occurring RLN is synthesized as a single-chain pro-RLN consisting of a receptor binding B-chain on the N-terminus, an A-chain on the C-terminus that forms disulfide bridges with B-chain to improve its stability, and a connecting C-chain in between. Processing of the pro-RLN to RLN occurs in vivo through the endoproteolytic cleavage of the C-peptide. However, delivering the A and B-chain peptides separately often leads to reduced protein stability and assembly challenges.37 Therefore, we retained the original mRNA sequence encoding the pro-RLN (Fig. 4a). As the B-chain’s two receptor binding sites are crucial for RLN function, we added the CBD peptides adjacent to the A-chain and close to the C terminus. There is low homology between human and mouse RLN and mouse RLN 1 exhibits similar folding and functionality to human RLN 2 which is currently being studied in clinical trials.38 We therefore designed mouse and hamster RLN 1 fusion proteins tailored to our animal models. To preserve the structure of both RLN and CBD, we incorporated a flexible GGGS linker between the CBD and A chain. Pseudo-uridine-modified mRNAs encoding the 12 fusion proteins were prepared using in vitro transcription (Fig. S18). These mRNAs were then formulated with the previously screened ATRA LNPs. Cryo-electron microscopy (Cryo-EM) images confirmed the presence of uniformly solid spherical structures of the resultant RLN-CBD mRNA (mRLN-CBD) LNP formulations (Fig. 4c). The expression of LNP delivered mRLN-CBD was confirmed through IF analysis of the fixed cells and enzyme-linked immunosorbent assay (ELISA) of the supernatant. All fusion peptides exhibited similar expression levels, which were comparable to or slightly lower than that of the unmodified RLN peptide (Fig. S19). To assess the binding capability of the fusion protein with collagen, we conducted a sandwich ELISA study. We collected supernatant from mRNA treated cells to quantify the concentration of the secreted protein, and evaluated the binding of the flag-tagged fusion protein to a collagen-coated plate with anti-flag tag antibodies (Fig. 4d). The results demonstrated that the addition of CBD Pep K to RLN led to strong and versatile binding to ECM proteins (Fig. 4e and S20).14 Pep K is derived from collagen binding domains found in placenta growth factor-2 (PLGF-2123−144), and was selected as the CBD domain candidate for the remainder of our study.
RLN-PLGF1 mRNA delivered by ATRA LNPs showed improved retention and comparable activity in fibrotic livers
To further augment the collagen binding capability of our RLN-CBD fusion protein, we made an additional fusion protein with an extended PLGF motifs (increased from 1 unit to 3 units). We termed these fusion proteins RLN-PLGF1 and RLN-PLGF3 respectively. We then studied the properties of the RLN-PLGF modified fusion proteins compared to two controls: an unmodified RLN and an RLN fused with an Fc domain (RLN-Fc) known to prolong systemic circulation (Fig. 5a). Molecular models of three fusion proteins were predicted with Alphafold2 using the crystal structure of human RLN-2 peptide as a template. The modeling results suggested that amino acid interaction between RLN, the Fc receptor and the CBD PLGF domains were minimal. Molecular dynamics simulations using all-atom force field further confirmed minimal interaction of RLN-PLGF1 and RLN-Fc outside the receptor binding domain of RLN, while RLN-PLGF3 showed significant interactions between the RLN and CBD domain with 8 hydrogen bonds observed that could potentially impact the folding of RLN and PLGF (Fig. 5b, Video S3-S5).
mRNA encoding for the four proteins was encapsulated (separately) in ATRA LNPs and delivered to HSCs (Fig. S21). Interestingly, results revealed that although the expression of RLN-PLGF3 could be detected through IF staining, the level of protein secretion was significantly lower compared to the other fusion proteins (Fig. 5c and S22). This observation is consistent with the molecular simulation and suggests that the interaction between PLGF and RLN may hinder the protein folding or protein secretion.
To assess the collagen binding capabilities, we performed the sandwich ELISA assay, which showed that RLN-PLGF1 demonstrated improved binding to collagen compared to RLN-PLGF3, RLN and RLN-Fc. We then purified RLN, RLN-PLGF1 and RLN-Fc, and determined their Kd values against collagen II using surface plasmon resonance (SPR). Results showed a relatively high binding affinity with ~ 20 nM of Kd value for the RLN-PLGF1 fusion protein. In contrast, unmodified RLN and RLN-Fc did not exhibit significant binding to collagen II (Fig. 5e).
To explore the protein retention in vivo, we delivered 1.5 mg/kg of each of the four mRNAs to CCl4-treated mice using ATRA LNPs (1.5 mg/kg) (Fig. 5f). RLN-PLGF1, but not RLN-PLGF3 or RLN-Fc, improved the accumulation of RLN within the liver (Fig. 5g and h). The AUC of RLN in mRLN-PLGF1 group was approximately 2 times higher than mRLN-Fc and 3 times higher than unmodified RLN (Fig. 5g and h). Notably, RLN-Fc exhibited prolonged systemic circulation, while minimal RLN-PLGF1 was detected in the blood during sample collection (Fig. 5g and h). Three days post injection, over 80% of RLN-PLGF1 was retained in the liver, whereas approximately 80% of RLN-Fc were circulated in the bloodstream (Fig. 5i). Both free RLN and RLN-Fc showed significantly higher levels of inflammatory cytokines (e.g. IL-6, IFN-γ) within 6 days of injection compared to RLN-PLGF1, confirming systemic toxicity was reduced using the RLN-PLGF1 modality (Fig. 5j, k and Fig. S23).
The biological activities of the recombinant proteins were measured using a cAMP activation assay. All mRNA encoding fusion proteins activated cAMP, with RLN-PLGF3 showing slightly lower activation compared to other treatment groups (Fig. 5l). The fusion proteins were able to inhibit α-SMA and TGF-β expression, key proteins regulated by RLN to control fibroblast activation, at both the mRNA and protein levels. However, RLN-PLGF3 showed the lowest activity in inhibiting TGF-β expression and almost no inhibition of α-SMA at the protein level. These findings support our modeling and simulation data which suggested that RLN-PLGF3 would have compromised the biological activities due to intramolecular interactions (Fig. 5m-o).
RLN acts through the RXFP1 receptor.38 MST assays were performed to examine if RLN to RXFP1 binding were negatively impacted using the collagen anchored RLN-PLGF1 (Fig. 5p and S24). Cell lysates from 293F cells containing free RLN-His-tag protein and RLN-PLGF1-His-tag protein were incubated with collagen IV at the saturation concentration. RXFP1+/RXFP1− cells were then incubated with free and collagen treated RLN-His-tag protein and RLN-PLGF1-His-tag protein. The addition of collagen did not significantly affect the binding of RLN-PLGF1 to RXFP1+ cells. No significant binding was observed for RXFP1− cells (Fig. S24). Collectively, these findings demonstrate that the fusion of RLN with 1 unit of the PLGF domain facilitates collagen anchoring whilst maintaining RLN function.
mRLN-PLGF 1 in ATRA LNPs reduces fibrosis and fatty liver in a CCl4-treated fibrosis and MCD models
We assessed the anti-fibrosis effect of the FORT strategy in CCl4-induced liver fibrosis mouse models (Fig. 6a). Four doses of ATRA LNPs containing different mRNA constructs (encoding RLN, RLN-PLGF1, RLN-PLGF3 and RLN-Fc) or empty LNPs were i.v. administrated to CCl4-treated mouse. As a positive control, we also orally administered obeticholic acid (OCA), a clinically investigated small molecule for treating NASH (Fig. 6a).27 OCA reduced liver index and collagen deposition (Fig. 6b-f), leading to a decrease in NASH severity. However, its effect on serum alanine aminotransferase (ALT) and aspartate transaminase (AST) was minimal. This is consistent with clinical observations in humans.27
ATRA LNPs containing mRLN moderately reduced liver fibrosis index and AST/ALT levels. In contrast, mRLN-Fc LNPs, which extended the systemic circulation, showed enhanced therapeutic improvement compared to mRLN LNPs. However, mRLN-Fc LNPs showed systemic toxicity with significant weight loss (Fig. S25). Notably, treatment with mRLN-PLGF1 LNPs demonstrated the most significant benefits in reversing liver fibrosis. It led to a ~ 13% decrease in liver index, normalized AST/ALT, 6.4- fold reduction in α-SMA expression, 4.3- and 6.7- fold decrease in Masson’s trichrome and Sirius red staining, more pronounced than the positive control OCA group. H&E staining also revealed reduced liver damage (Fig. 6b-f). In contrast, mRLN-PLGF3 LNPs failed to show significant improvement of liver damage which supports our earlier in vitro data (Fig. S25).
The anti-fibrosis effect of the FORT strategy using therapeutic RLN was further examined in mice fed on an MCD diet. The model is accompanied with fat accumulation and liver fibrosis. We extended the dosing intervals to five days to challenge the retention capacity of the strategy (Fig. 6i). As in the CCl4-induced liver fibrosis, mRLN-PLGF1 demonstrated enhanced therapeutic effects compared to the unmodified mRLN LNP (Fig. 6j-p). In fact, the reduction in collagen coverage in the liver was 2- fold greater in the mRLN-PLGF1 group than in mRLN group, as evidenced by Sirius red and Masson’s trichrome staining (Fig. 6l and m). In the mRLN-PLGF1 group, significantly lower levels of liver index, AST/ALT levels were observed (Fig. 6j and k). Interestingly, Oil red O staining revealed that mRLN-PLGF1 LNPs effectively cleared lipid droplets in the MCD-induced mouse model. We conducted gene expression analysis in treated mice to identify mechanisms involved in this change. Our results showed a more pronounced reduction (~ 2-10- fold) in inflammatory cytokines (e.g. IL-1β, IL-6) productions in mice treated with mRLN-PLGF1 LNPs group compared to mRLN LNPs (Fig. S26). Additionally, there was a slight decrease in expression of fatty acid uptake genes (Fabp1, Cd36, Lipin1) and lipogenesis genes (Srebp1c, Fasn, Dgat2), along with upregulation of genes involved in lipid oxidation (Acot1). These findings suggest that ATRA LNPs formulated mRLN-PLGF1 have the potential to remodel the NASH microenvironment by affecting both fibrotic and lipid biosynthesis pathways.
Low-dose combination of mRLN-PLGF 1 and mIL-10-PLGF1 LNP leads to outstanding performance in hamster models with NASH
To further investigate the potential of FORT strategy in clinical application, we applied it to a more clinically relevant NASH model using hamsters fed with CDHFD diet. This model closely mimics the metabolic profile and pathogenesis of human (Fig. 7a, Fig. S2). In addition to mRLN-PLGF1, we introduced IL-10-PLGF1, another CBD-based fusion protein, by fusing PLGF peptide on the C terminus (Fig. 7b). Adding PLGF1 on IL-10 also improved the binding to ECM proteins (Fig. S20). Both mRLN-PLGF1 and mIL-10-PLGF1 were formulated with ATRA LNPs and administered through jugular vein. We extended the therapeutic intervals to 6 d per dose. Significant echo signal reduction was observed using ultrasound imaging within 18 days when treated with either mono or combined therapy (Fig. 7c and S27). Remarkably, combination of mRLN-PLGF1 and mIL-10-PLGF1 LNP substantially ameliorated liver fibrosis and inflammation (Fig. 7c-k). This was characterized by the reduced and more homogenous echo intensity, as well as the lower levels of ALT/AST (Fig. 7e) and TC/TG compared to the sham group (Fig. 7d and k). Interestingly, mIL-10-PLGF1 LNPs significantly reduced TC/TG levels in the circulation, while mRLN-PLGF1 was more effective in downregulating liver fat and fibrosis, suggesting a potential synergism between the two regimens. After combo therapy, collagen disposition in liver was similar to the WT group (Fig. 7f and g). Combo therapy almost eliminated the accumulation of lipid droplets (Fig. 7h and i). We further investigated the genes associated with fibrosis and lipid metabolism. mRLN-PLGF1 LNP monotherapy induced a more substantial reduction of pro-fibrogenic factors, while the combo therapy fell in between, with ~ 5.7- fold, ~ 12.8- fold, ~ 8.0- fold downregulation of TGF-β, α-SMA, COL1α1, along with significant upregulation of MMPs, when compared to the sham group (Fig. 7l). mRLN-PLGF1 showed slight inhibition of lipogenesis genes (SCD1, SREBP1C), consistent with those observed in the MCD models. In contrast, mIL-10-PLGF1 treatment led to ~ 3.0-, ~ 7.0- and ~ 3.6- fold decrease of these genes (Fig. S27). The combination of both resulted in decreased lipid synthesis (Fig. 7l). Notably, promotion of fatty acid β-oxidation was observed in both therapies (Fig. 7l). These results demonstrate the feasibility of applying multiple FORT proteins to achieve synergistic effects and facilitate recovering of NASH. Moreover, neither of the proposed mRNA therapies induced histological abnormalities in major organs or caused significant changes in body weight when compared with sham treatment, suggesting negligible systemic toxicity, low immunogenicity or immunosuppression (Fig. S28).