RNA Nanotherapeutics with Fibrosis Overexpression and Retention (FORT) for NASH Treatment

RNA Nanotherapeutics with Fibrosis Overexpression and Retention (FORT) for NASH Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article RNA Nanotherapeutics with Fibrosis Overexpression and Retention (FORT) for NASH Treatment Lei Miao, Xinzhu Shan, Zhiqiang Zhao, Pingping Lai, Yuxiu Liu, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3746897/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Fibrotic diseases, like non-alcoholic steatohepatitis (NASH), pose challenges for targeted delivery and retention of therapeutic proteins due to increased extracellular matrix (ECM) deposition. Here we present a new approach to treat fibrotic diseases, termed “Fibrosis overexpression and retention (FORT)”. In this two-step strategy, we design 1) a retinoid derivative lipid nanoparticle (LNP) to enable specific mRNA overexpression in hepatic stellate cells, and 2) mRNA modifications which facilitate anchoring of therapeutic proteins in the fibrotic ECM. LNPs containing carboxyl retinoid derivatives, as opposed to alcohol or ester retinoid derivatives, effectively delivered mRNA, resulting in more than 10- fold enhancement of protein expression within the fibrotic liver. The carboxyl retinoid rearrangement on the LNP surface improved protein binding, sprouting, and membrane fusion. Therapeutic relaxin fusion proteins were then engineered with an endogenous collagen-binding domain. These fusion proteins exhibited increased retention in fibrotic lesions and reduced systemic side effects. In vivo , fibrosis-targeting LNPs encoding for mRNA fusion proteins demonstrated superior therapeutic efficacy in three clinically relevant NASH mouse models. This approach holds promise in chronic fibrotic diseases that are unsuited for direct injections of recombinant proteins. Biological sciences/Biotechnology/Biomaterials/Drug delivery Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles lipid nanoparticles carboxyl retinoids collagen binding domain relaxin NASH Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Non-alcoholic steatohepatitis (NASH) is a chronic liver inflammation that can progress to fibrosis and cancer, affecting approximately 5% of the population, causing significant mortality worldwide. 1 , 2 Currently, there are limited therapies for the treatment of NASH due to challenges with delivering to, and treating, the fibrotic microenvironment in the liver. 3 Inspired by the success of glucagon-like peptide 1 (GLP-1) in diabetes treating, protein-based therapies hold promise for NASH treatment. 4 , 5 In animal models of chronic liver injury, the protein relaxin (RLN) has shown benefits. 6 – 8 RLN interacts with the Relaxin Family Peptide Receptor 1 (RXFP 1 ) and inhibits the TGF-β pathway, which is implicated in fibrosis. 9 Recombinant RLNs are currently undergoing clinical trials for treating heart fibrosis. 8 Other proteins, such as anti-inflammatory cytokines like IL-10, have also been reported to protect mice from liver fibrosis by counterbalancing hyperactive immune responses. 10 However, these protein therapies are challenging to translate to the clinics due to limited stability, rapid clearance, and lack of organ targeting specificity. Strategies to extend protein circulation half-life have been successful for some clinically relevant therapeutic proteins (i.e., IL-2, GLP-1 and etc). Approaches have included protein modifications to introduce the Fc fragment of IgG 8 , PEGylation 11 , and the addition of long chain alkanes. 12 Yet, this may not be sufficient to enhance drug concentration at the lesion site and could potentially increase systemic toxicity. In parallel, the design of fusion proteins with specific binding domains has been shown to help anchor protein therapeutics in the lesions. 13 – 15 Unfortunately, the administration of these fusion proteins is limited to local injections in easily accessible disease models, and their manufacturing can be complex and requires further optimization. Therefore, developing strategies for targeted delivery of therapeutic anti-fibrosis proteins and prolonging their retention in fibrotic lesions are critical for NASH treatment. Lipid nanoparticle (LNP) mRNA therapies have shown enormous promise for expression of nucleic acids encoding for target therapeutic proteins. 16 For instance, the RNA drug Onpattro is clinically approved for treating transthyretin mediated amyloidosis and highlights the therapeutic potential of LNPs for liver diseases. LNPs often preferentially deliver nucleic acid cargo to the liver. This is thought to be due, in part, to adsorption of ApoE proteins to the LNP surfaces, and targeting other organs through chemical modifications to LNPs is an expanding research area. 17 , 18 Although the liver is considered a relatively accessible organ for mRNA LNPs in healthy patients, it becomes less amenable to therapeutic intervention during fibrosis. Hepatic stellate cells (HSCs), residing between liver sinusoidal endothelial cells (LSECs) and hepatocytes, become activated during fibrogensis. 19 , 20 These activated HSCs (aHSCs) deposit excessive amount of extracellular matrix (ECM) proteins in fibrotic lesions. This exacerbates inflammation, contributes to capillarization and closure of fenestrae, thus restricting drug delivery to the liver. 21 The NASH microenvironment is therefore exceptionally challenging for therapeutic delivery of mRNA LNPs. Here, we present a new strategy for delivering therapeutic proteins to hepatic NASH lesions via mRNA LNPs in a process we term “Fibrosis overexpression and retention (FORT)”. Briefly, we designed a new LNP which incorporates retinoid derivative ligands to facilitate targeted delivery to HSCs. Retinoid derivatives have previously been shown to facilitate fibrotic accumulation of therapeutic regimens due to “attach” to retinol-binding protein-4 (RBP-4). Among the analogues tested, carboxylic retinoids, not conventional alcoholic or ester-derived retinoids have shown improved mRNA expression in fibrotic livers. Mechanistic investigations revealed that the carboxylic retinoids rearranged on the outer shell of LNPs during mRNA encapsulation, resulting in improved binding to RBP-4, enhanced internalization, sprouting, and endosomal escape. We use these LNPs to deliver mRNA encoding for engineered proteins which co-express an endogenous peptide domain to “anchor” proteins into the fibrotic ECM. Using this approach, our lead ligand candidate (all-trans retinoic acid, ATRA) demonstrated a ~ 10- fold increase of mRNA expression in fibrotic livers compared to commercial ALC-0315 formulations. We used this LNP to deliver mRNA encoding for an anti-fibrotic RLN protein fused to an optimized collagen binding domain (CBD) from placenta growth factor (PLGF). We demonstrate 80% retention of the fusion protein in the liver. We further demonstrate therapeutic reduction in fibrosis in three clinically relevant NASH models. We believe the FORT approach could be widely applicable to the delivery and anchoring of therapeutic proteins to less accessible fibrotic microenvironment. RESULTS 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. 2 a-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 (CCl 4 ) and a NASH hamster model induced by 10-week choline deficient high fat diet (CDHFD) treatment (Fig. 2 b, 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 strategy 22 , 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. 2 d), 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. 2 d). 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. 2 d). 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. 2 e 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 CCl 4 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. 2 f, g, Fig. S5 ). This observation aligned with the in vitro transfection study conducted in LX-2 cells (Fig. 2 e), 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 CCl 4 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. 2 h). Over 95% of mRNA was expressed in liver rather than other organs (Fig. 2 i, 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 CCl 4 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. 2 j, 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 CCl 4 treated tdTomato reporter mice (Fig. 3 a). 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. 3 a). 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. 3 b, Fig. S9 ). This result aligns with the in vitro study and suggests that ATRA LNPs tend to accumulate in the fibrotic area (Fig. 3 c). 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. 3 d). 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. 3 e, 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. 3 f 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. 3 p). 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 (D 2 O) 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. 3 h) 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. 3 i ) , 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. 3 j, 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. 3 k). 31 The ratio of DOPC and DPPC was tuned to match the saturation levels of the endosomal membranes from HSCs and hepatocytes (Fig. 3 l). 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. 3 m, n, Video S1 ). However, in the rigid membrane mimicking HSC endosomes, almost no fusion events were observed for ALC-0315 LNPs (Fig. 3 m, n, Video S2 ). Nevertheless, ATRA LNPs exhibited over 50% fusion at the end point (Fig. 3 m), with faster diffusion compared to other LNP treated membranes ( Fig. 3 o). 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. 3 p). 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. 4 a ) . 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. 4 b). 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. 4 a). 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. 4 c). 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. 4 d). The results demonstrated that the addition of CBD Pep K to RLN led to strong and versatile binding to ECM proteins (Fig. 4 e and S20). 14 Pep K is derived from collagen binding domains found in placenta growth factor-2 (PLGF-2 123−144 ), and was selected as the CBD domain candidate for the remainder of our study. RLN-PLGF 1 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-PLGF 1 and RLN-PLGF 3 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. 5 a ) . 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-PLGF 1 and RLN-Fc outside the receptor binding domain of RLN, while RLN-PLGF 3 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. 5 b, 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-PLGF 3 could be detected through IF staining, the level of protein secretion was significantly lower compared to the other fusion proteins (Fig. 5 c 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-PLGF 1 demonstrated improved binding to collagen compared to RLN-PLGF 3 , RLN and RLN-Fc. We then purified RLN, RLN-PLGF 1 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-PLGF 1 fusion protein. In contrast, unmodified RLN and RLN-Fc did not exhibit significant binding to collagen II (Fig. 5 e). To explore the protein retention in vivo , we delivered 1.5 mg/kg of each of the four mRNAs to CCl 4 -treated mice using ATRA LNPs (1.5 mg/kg) (Fig. 5 f). RLN-PLGF 1 , but not RLN-PLGF 3 or RLN-Fc, improved the accumulation of RLN within the liver (Fig. 5 g and h ). The AUC of RLN in mRLN-PLGF 1 group was approximately 2 times higher than mRLN-Fc and 3 times higher than unmodified RLN (Fig. 5 g and h ). Notably, RLN-Fc exhibited prolonged systemic circulation, while minimal RLN-PLGF 1 was detected in the blood during sample collection (Fig. 5 g and h ). Three days post injection, over 80% of RLN-PLGF 1 was retained in the liver, whereas approximately 80% of RLN-Fc were circulated in the bloodstream (Fig. 5 i). 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-PLGF 1, confirming systemic toxicity was reduced using the RLN-PLGF 1 modality (Fig. 5 j, 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-PLGF 3 showing slightly lower activation compared to other treatment groups (Fig. 5 l). 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-PLGF 3 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-PLGF 3 would have compromised the biological activities due to intramolecular interactions (Fig. 5 m-o). RLN acts through the RXFP 1 receptor. 38 MST assays were performed to examine if RLN to RXFP 1 binding were negatively impacted using the collagen anchored RLN-PLGF 1 (Fig. 5 p and S24). Cell lysates from 293F cells containing free RLN-His-tag protein and RLN-PLGF 1 -His-tag protein were incubated with collagen IV at the saturation concentration. RXFP 1 + /RXFP 1 − cells were then incubated with free and collagen treated RLN-His-tag protein and RLN-PLGF 1 -His-tag protein. The addition of collagen did not significantly affect the binding of RLN-PLGF 1 to RXFP 1 + cells. No significant binding was observed for RXFP 1 − 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 CCl 4 -treated fibrosis and MCD models We assessed the anti-fibrosis effect of the FORT strategy in CCl 4 -induced liver fibrosis mouse models (Fig. 6 a). Four doses of ATRA LNPs containing different mRNA constructs (encoding RLN, RLN-PLGF 1 , RLN-PLGF 3 and RLN-Fc) or empty LNPs were i.v. administrated to CCl 4 -treated mouse. As a positive control, we also orally administered obeticholic acid (OCA), a clinically investigated small molecule for treating NASH (Fig. 6 a). 27 OCA reduced liver index and collagen deposition (Fig. 6 b-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-PLGF 1 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. 6 b-f ) . In contrast, mRLN-PLGF 3 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. 6 i ) . As in the CCl 4 -induced liver fibrosis, mRLN-PLGF 1 demonstrated enhanced therapeutic effects compared to the unmodified mRLN LNP (Fig. 6 j-p ) . In fact, the reduction in collagen coverage in the liver was 2- fold greater in the mRLN-PLGF 1 group than in mRLN group, as evidenced by Sirius red and Masson’s trichrome staining (Fig. 6 l and m) . In the mRLN-PLGF 1 group, significantly lower levels of liver index, AST/ALT levels were observed ( Fig. 6 j and k) . Interestingly, Oil red O staining revealed that mRLN-PLGF 1 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-PLGF 1 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-PLGF 1 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-PLGF 1 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. 7 a, Fig. S2 ). In addition to mRLN-PLGF 1 , we introduced IL-10-PLGF 1 , another CBD-based fusion protein, by fusing PLGF peptide on the C terminus ( Fig. 7 b ) . Adding PLGF 1 on IL-10 also improved the binding to ECM proteins ( Fig. S20 ). Both mRLN-PLGF 1 and mIL-10-PLGF 1 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. 7 c and S27). Remarkably, combination of mRLN-PLGF 1 and mIL-10-PLGF 1 LNP substantially ameliorated liver fibrosis and inflammation (Fig. 7 c-k). This was characterized by the reduced and more homogenous echo intensity, as well as the lower levels of ALT/AST ( Fig. 7 e ) and TC/TG compared to the sham group ( Fig. 7 d and k) . Interestingly, mIL-10-PLGF 1 LNPs significantly reduced TC/TG levels in the circulation, while mRLN-PLGF 1 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. 7 f and g) . Combo therapy almost eliminated the accumulation of lipid droplets ( Fig. 7 h and i) . We further investigated the genes associated with fibrosis and lipid metabolism. mRLN-PLGF 1 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. 7 l ) . mRLN-PLGF 1 showed slight inhibition of lipogenesis genes ( SCD1 , SREBP1C ), consistent with those observed in the MCD models. In contrast, mIL-10-PLGF 1 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. 7 l ) . Notably, promotion of fatty acid β-oxidation was observed in both therapies ( Fig. 7 l ) . 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 ). DICUSSIONS Fibrotic diseases are a leading cause of morbidity and mortality worldwide. Here we present a new approach to treating fibrotic diseases, termed “Fibrosis overexpression and retention (FORT)”. In this two-part strategy, we design 1) retinoic acid LNPs which distributed retinoids on the surface of the particles, facilitating RBP-4 mediated endocytosis and sprouting/fusion-mediated endosome escape in HSCs, and 2) modified mRNAs which encode for CBDs allowing us to anchor expressed therapeutic fusion proteins to fibrotic ECM. We believe this is the first time that fibrotic lesions have been targeted and treated in this way. This approach offers advantages over current modalities as it confines the therapeutic protein to the disease region, extending the duration of action and minimizing systemic exposure. Fibrotic liver diseases such as NASH and cirrhosis are characterized by capillary base thickening and ECM accumulation. These biophysical changes increase difficulty for therapeutic delivery. In our study, we found commercial LNP formulations were ~ 10- fold less effective at delivering mRNA to NASH animal models. We proposed the physical incorporation of FDA-approved molecules to enhance mRNA delivery to HSCs in fibrotic livers. Retinoid derivatives were chosen due to their high no-observed-adverse-effect level (NOAEL) in patients and their established suitability for targeting HSCs. Although targeted moieties such as antibodies, nanobodies and aptamers have been widely explored to improve HSC targeting, they often present challenges in terms of scaling up complexity and batch-to batch variability. 39 In contrast, the physical incorporation 40 of hydrophobic moieties into lipid bilayers presents a simple targeting approach with ease of manufacturing. This approach also facilitates incorporation of small molecules over a wide dose range. Our study focused on exploring the structure-activity relationship of the retinoid derivatives by physically encapsulating various moieties within LNPs. Surprisingly, conventional retinol or uncharged esters did not exhibit effective targeting to HSCs, while carboxylic acid derivatives demonstrated improved targeting. Mechanistic studies suggested that these derivatives may induce a spatial reorganization of lipids when condensed with mRNA, affecting RBP-4-mediated cell uptake. Additionally, we discovered that the endosomal membrane of HSCs is more rigid than that of hepatocytes. ATRA LNPs with enhanced sprouting and fusion rate demonstrated superior fusion with HSC-endosome mimicking membranes, potentially promoting RNA release. To enhance therapeutic efficacy, new strategies enabling protein retention without compromising biological activity are required. Previous studies have demonstrated that binding therapeutic antibodies or proteins to collagen can enhance local accumulation. However, the applications have been limited by delivery challenges. In our study, we delivered designed mRNA encoding for therapeutic proteins fused to CBD to facilitate retention in the NASH fibrotic lesion. We selected a CBD motif from endogenous protein domains, as opposed to peptide sequences obtained from display techniques, since the endogenous CBDs offer advantages such as lower immunogenicity and higher specificity. Notably, we found that the addition of multiple CBDs does not further enhance binding or efficacy. In contrast, it might induce mis-folding, compromising therapeutic efficacy. Therefore, we chose to combine therapeutic proteins with a single CBD, identified from PLGF. RLN is a promising therapeutic protein for treatment of fibrosis currently in clinical trials. 41 However, clinical failures of RLN peptides have been primarily attributed to undesirable pharmacokinetics. 42 Additionally, RLN lacks an intrinsic CBD to facilitate its interaction with the ECM. We demonstrate that our modified RLN-PLGF 1 protein improved fibrotic liver accumulation of RLN protein and increased anti-fibrosis activity in three NASH models. We also extended the use of FORT strategy to other secretable proteins such as IL-10. In conclusion, we have developed a new type of mRNA LNP therapy for the treatment of fibrosis, termed FORT. By incorporating an FDA-approved retinoic acid in LNPs, the expression of RNA therapeutics in fibrotic liver was significantly improved. Further, the addition of an endogenous CBD domain from PLGF to mRNA sequences generates a fusion protein with improved retention in fibrotic lesions. Delivery of therapeutic proteins using these strategies ameliorate fibrosis in animal models. We believe this approach can be broadly applied to other chronic inflammatory diseases that are not amenable to direct injection but can be targeted using LNPs. Materials and Methods Materials All the ionizable lipids containing DLin-MC3-DMA (MC3), 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) and 1-octylnonyl 8-(2-hydroxyethyl)6-0x0-(undecyloxy)hexyl amino-octanoate (SM-102) were purchased from Avanti Polar Lipids, Inc. Helper lipids containing cholesterol, 1,2-Dioctadecanoyl-sn-glycero-3-phophocholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) were bought from A.V.T. Pharmaceutical Tech Co., Ltd. Small-molecular retinoids containing retinol, 4-keto-retinol, all-trans-retinoic acid (ATRA), 13-cis retinoic acid (13-CRA), Acitretin (A-VA), were purchased from Sigma-Aldrich, 9-cis retinoic acid (9-CRA), fenretinide and acetyl-retinol were purchased from Macklin Co., Ltd, bexarotene and tamibarotene were obtained from MedChemExpress Co., Ltd. Firefly luciferase mRNA (mLuc) was provided by Proxybio. Collagen I, II was purchased from Sino Biological and Collagen IV from Sigma. Human and mouse RBP-4 were purchased from Sino Biological Inc. Purified RLN, RLN-PLGF 1 , RLN-Fc and cell lysates containing RLN and RLN-PLGF 1 were provided by KeyMed Biosciences. The small-molecular uptake inhibitors and carbon tetrachloride were purchased from Macklin Co., Ltd. Methionine-choline-deficient (MCD) diet and choline-deficient high-fat diet (CDHFD, 45% kcal) was purchased from Dytes Inc. All the plasmid sequences were provided by Genscript Co.,Ltd. Primers and siRNA were from Tsingke Biotechnology Co.,Ltd. All cell lines and fetal bovine serum (FBS) were purchase from Procell Life Science & Technology Co.,Ltd. Other reagents for basal culture were bought from Meilunbio Co., Ltd. ELISA kits for mouse IL-6, TNF-α, IL-1β and IFN-γ were purchased from Solarbio Science & Technology Co.,Ltd., mouse RLN-1 from Boster Bio and cAMP from Elabscience Biotechnology Co.,Ltd. Methods 1. mRNA synthesis mRLN, mRLN-PLGF 1 , mRLN-Fc, mIL-10-PLGF 1 , mLuc and mCre were synthesized using T7 polymerase mediated in vitro transcription (IVT) system from linearized pUC57 plasmid vectors containing T7 promoter, 5ʹ and 3ʹ untranslated regions (UTRs) and a poly A tail (100 nt). The clean-cap AG 5’ capping (Cap 1) and 1-methylpsuedo-uridine UTP were added to the transcription reaction. The uridine-5’triphosphate (UTP) was fully replaced with 1-methylpsudeo-uridine UTP to improve protein translation and minimize immunogenicity generated from synthesized mRNA. IVT reactions were conducted according to the manufacturers’ protocols (Hongene Biotech Inc., China). The mRNAs encoding fusion proteins containing 11 different CBD domains (i.e. RLN and IL-10) for initial screening were synthesized from linearized pUC57 plasmid vectors with the absence of poly A sequence. Poly A were added after IVT reactions using Poly A Polymerase Tailing Kit (Beyotime Biotechnology). The CDS sequences of mRNAs used in the current study were listed in Table S8 . Purity of the linearized plasmids and synthesized mRNAs were validated by gel electrophoresis ( Fig. S20 and S23 ). 2. Lipid nanoparticle preparation and characterization LNPs were prepared either by hand mixing or microfluidic mixing as previously described. Briefly, an aqueous solution of the mRNA and an ethanolic solution of the lipid components were mixed at a ratio of 3:1, respectively. The ethanol phase consists of ionizable ALC-0315 (Avanti), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC, AVT, China), cholesterol (AVT, China) and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000, AVT, China) and a series of FDA approved retinoids at the predetermined molar ratio ( Table S1 ). The aqueous phase was prepared in 10 mM citrate buffer containing 0.14 µg/µL mRNA. After mixing, the obtained LNPs were dialyzed against 1× PBS in dialysis bag at 4°C overnight. mRNA concentration and encapsulation efficiency of LNP were measured using Quant-it RiboGreen RNA assay (Invitrogen). The hydrodynamic diameter and zeta potential were measured by dynamic light scattering (Zetasizer Nano ZSP, Malvern). The morphology of LNPs were characterized by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). For cryo-EM, the prepared LNP were dialyzed in 20 mM Tris (pH 7.4) containing 8% sucrose 4°C overnight, and then concentrated to 0.5 mg/ml total RNA by ultrafiltration. Cryo-EM image was acquired using Themis 300 (Thermo Fisher Scientific). To evaluate the plasma stability of LNPs, ALC-0315, retinol and ATRA LNPs were incubated in DMEM culture medium (pH 7.4) containing 10% FBS for 12 h at 37 ℃. The particle sizes of LNPs were measured by a Zetasizer at predetermined time points. 3. Small angle neutron scattering (SANS) In this work, SANS was performed on the Small Angle Neutron Scattering (SANS) instrument at China Spallation Neutron Source (CSNS). 30 The incident neutrons with wavelength of 1–10Å were defined by a double-disc bandwidth chopper, which is collimated to the sample by a pair of apertures. The experiment used the sample to detector distance of 4 m and a sample aperture of 6 mm. The 1m square detector array composed of 120 linear He-3 gas tubes with the diameter of 8 mm, which covers the Q-range between 0.01Å-1 and 1Å-1. The presented data correspond to ~ 120 min of data collection time for each sample (@140kW). For all SANS data, background signals from the solvent, sample cell and the instrument were subtracted by separate runs to measure their scattering contributions. Neutron data were normalized and corrected for transmission and detector efficiency, and set to absolute units. Modeling and simulation details are provided in Supplementary Methods . 4. Microscale thermophoresis (MST) assay The binding affinities between RBP-4 to free retinoids, empty and mRNA loaded LNP were measured using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany). Firstly, RBP-4 was adjusted to a concentration of 10 µM and labeled with MonolithTM NT.115 protein Labeling Kit RED-NHS (Nanotemper Technologies, Germany), following the manufacturer’s protocols. The labeled RBP-4 was then purified by gel filtration and diluted to a 250 nM solution using PBS containing 0.05% Tween 20 (PBS-T), ensuring that the fluorescent intensity of RBP-4 during the MST assay was approximately 500 response units (RU). LNPs with an initial concentration of 1 mg/mL (total lipid) were serially diluted (16-point, 1:1 dilution in PBS-T) and mixed in equal volume with the pre-diluted RBP-4. The mixture was incubated for 1h at room temperature. After incubation, the samples were loaded into premium treated capillaries and measured using the NanoTemper Monolith NT.115 instrument. The dissociation equilibrium constant (KD) values were fitted according to the KD Model (1:1 binding mode), by the NanoTemper Monolith affinity software MO. Affinity Analysis v2.3 (NanoTemper Tecchnologies, Germany). The binding affinity measurement between the RLN fusion protein (with or without the presence of collagen) and surface receptor RXFP 1 was carried out using crude cell lysates, and measured by NanoTemper Monolith NT.115 instrument following similar protocols as described above. Details are provided in the Supplementary Materials . 5. In vitro collagen binding assay Cells were seeded in 6-well plates at a density of ~ 5×10 5 cells overnight and treated with mRNA-LNP. After 24 h of incubation, the cell culture supernatant was collected and incubated in 96-well ELISA plates that were pre-coated with collagen I, collagen II, collagen IV and BSA (10 µg/mL). The plates were then blocked with a solution of 2% BSA in PBS-T. Following a 1 h incubation at 37°C, the supernatant was removed, and the plates were washed and incubated with rabbit anti-Flag-tag antibody for 1 h at 37°C. After incubation, the plates were washed and incubated with HRP-conjugated goat anti-rabbit antibody for additional 1 h at room temperature. The collagen binding capacity was determined by measuring the absorbance of colorimetric TMB substrate, which reacts with HRP at 450 nm. 6. Surface plasmon resonance The SPR binding assays were performed using Biacore 8K + (Biacore, Cytiva). Recombinant collagen II (Sino Biological) was immobilized onto a CM5 sensor chip using standard amine coupling at 25°C. The reference flow cell was activated and subsequently blocked with BSA. The immobilization levels of collagen II were consistently around 3000 RU. To evaluate the binding affinity, various concentrations of RLN, RLN-PLGF 1 , RLN-Fc (6.25, 12.5, 25, 50, 100 and 200 µM) were injected into the channel. The binding assays involved subtracting the response observed in the reference flow cell containing BSA to account for non-specific binding. Regeneration of the sensor chip was achieved by performing extended washes with NaOH (5 mM) after each sample injection. The dissociation constants (KD) were determined by fitting the obtained data sets to a steady-state affinity model using the Biacore 8K Evaluation Software. 7. Molecular modeling and dynamics simulation of fusion proteins To predict the molecular models of all fusion proteins, we employed ColabFold V1.5.2 31 and run it in Google Colaboratory. We used the crystal structure (PDB: 6RLX) of human RLN-2 peptide 32 as a template to predict the RLN-PLGF 1 fusion protein. Among the generated models, the one with the highest pLDDT score (predicted local distance difference test) was selected as the best model for the further modeling and molecular dynamics experiments. For predicting RLN-PLGF 3 and RLN-Fc, the previously predicted RLN-PLGF 1 model and the crystal structure of human RLN-2 peptide (PDB:6RLX) were used as templates. Again, the models with the highest pLDDT score were selected as the best models for the subsequent molecular dynamic experiments. To carry out the molecular dynamics simulations, Groningen Machine for Chemical Simulations v2019.5 (GROMACS) was employed. The simulation runs lasted for 100 ns for RLN-PLGF 1 and 200 ns for RLN-PLGF 3 and RLN-Fc. The CHARMM27 all-atom force field 33 was used for these simulations. Each system was placed in a cubic water box consisting of the TIP3P water model, which was neutralized with counter ions. The system was initially minimized using the steepest descent algorithm through 1000 steps to eliminate any unfavorable contacts. Subsequently, the equilibration processes were conducted using constant number, volume (NVT), and temperature (NPT). The protein backbone was constrained, while the solvent molecules and counter ions were allowed to move freely. The NVT was carried out for 100 ps at 300K. The NPT was executed for 100 ps at 1 bar. The restraints on heavy atom bonds were imposed using the LINCS algorithm. 34 The Particle Mesh Ewald (PME) 35 method was used to calculate long-range electrostatic interactions, with a cutoff value of 10 Å for short-range interactions. Periodic boundary conditions were implemented to avoid edge effects. The coordinate data were saved with a time step of 2 fs for every 1 ps. Finally, Visual Molecular Dynamics (VMD) 36 and Pymol 37 were used for the evaluation of the results. 8. Endosome isolation, characterization and lipidomics Cells in culture were collected by centrifugation (850 g, 2 min, 4°C). Tissues were minced into small pieces, and subsequently homogenized using Dounce Homogenizer on ice. Cell lysis and organelles were then removed using lysosome enrichment kit according to the manufacturers’ protocol. Endosome was further isolated by density gradient centrifugation for 145,000 g, 2 h at 4°C. The collected endosomes were washed three times and characterized using western blotting western with Rab-5, the biomarkers of endosome (Cell Signaling Technology). The isolated organelles were gone through lipidomics ( Supplementary Methods ). 9. Cells culture and assays Cell lines were maintained in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Procell, China) and penicillin, streptomycin (Meilunbio, China). For activation of 3T3 cells, TGF-β1 was added at 10 ng/ml for 24 h. 9.1 In vitro transfection Cells were plated in white, clear-bottom 96-well plates at 4000 cells per well for LX-2 cells and 1×10 4 cells per well for primary hepatocytes. On the second day, mLuc LNP was added to cells at 0.1µg mRNA per well. After 24 h incubation, the transfection efficiency was measured by Firefly-Glo Luciferase Reporter Assay Kit (Yeasen Biotechnology Co., Ltd.) following the manufacturer’s protocol, using BioTek synergy H1 microplate reader. 9.2 In vitro cell uptake Primary hepatocytes and LX-2 stellate cells were plated in 96-well plates at a density of ~ 10 4 cells per well 24 h prior to the experiment. The cells were pre-incubated with small molecule endocytic inhibitors for 15 min. Small molecule endocytic inhibitors used in current study are listed below: cytochalasin D used for F-actin polymerization inhibition at 2.5µg/mL; methyl-β-cyclodextrin used for cholesterol/caveolae depletion at 2.5 mg/mL; nocodazole used for microtube inhibition at 5µg/mL; poly I used at the scavenger receptor inhibitor at 10 µg/mL; wortmannin used for inhibiting phosphoinositide pathway at 100 ng/mL and dynasore used for GTPase dynamin inhibition at 10 µg/mL. In addition, siRNA used for the knockdown of RBP-receptor, STRA6 was added at 200 nM 24 h at 37°C prior to LNP incubation. After pre-incubation with small molecules and siRNA, the supernatant was aspirated, washed twice with PBS. Then, 2.5 mol% BODIPY-lipid labeled LNPs (equivalent to 0.2 µg RNA per well) were added into each well of cells. After 2 h incubation at 37°C, the wells were washed 3 times with cold PBS and replaced with fresh media. The cellular uptake was determined by High Content Imaging and Analysis System (Cell Voyager CV8000, Yokogawa). Nucleus and lysosome were stained with Hoechst 33342 (1 µg/mL) and LysoTracker Red DND-99 (10000× dilute) at 10 min before imaging. Flow cytometric analysis was performed 24 h after LNPs treatment and the mean fluorescence intensity was applied for quantification. 9.3 Live-cell imaging of mRNA release Intracellular release of mRNA from LNPs was visualized using an inverted Zeiss LSM880 confocal microscope. Airyscan array detector unit (Carl Zeiss AG) was used to strengthen and visualize the cytosol mRNA signals within the live cells. Five % BODIPY-lipid and 100% Cy-3-UTP replaced mRNA were applied for tracing the intracellular behavior of LNP and mRNA. Cells were treated with the labeled LNP with mRNA at a concentration of 2 µg/mL and incubated for 2 hours at 37°C. Subsequently, the media were replaced with fresh media containing 1 µg/mL Hoechst 33342, and endosome release was observed using a 100× Plan-Neofluar 1.3 numerical aperture (NA) oil-immersion objective. For all experiments, the field of view (FOV) was set to 354, 25 µm × 354, 25 µm (full). The pinhole was set to 1 Airy Unit (AU) for all channels. The acquired imaging data were processed using Zen2.3 software (blue edition) for analysis. 10. Animal models All animal research was in compliance with ethical regulations approved by Peking University’s Institutional Animal Care and Use Committee. For models of CCl 4 -induced liver fibrosis, 8-week-old male C57BL/6 mice were intraperitoneal injected with CCl 4 /olive oil (7/13, v/v, 50 µL per mouse) three times a week for a total of 4 or 6 weeks. For NASH models of C57BL/6 mice and golden hamster, 8-week-old male animals were fed with MCD diet for 8 weeks or CDHFD diet for 10 weeks. Fibrosis or NASH level were monitored by measuring serum AST and ALT levels. 10.1 In vivo expression mLuc LNPs were i.v. administrated to healthy mice or mice with liver fibrosis (0.25 mg/kg). At 2, 6, 12 and 24 h after injection, bioluminescence imaging was performed using IVIS imaging system (Perkin Elmer). Hamsters with NASH were anesthetized by inhalation of isoflurane. A midline incision was made between the chin and sternum. The peripheral muscles were separated to expose the common carotid artery. LNPs at a dose of 0.25 mg/kg were injected through the carotid artery. Bioluminescence images were taken following the same process as described above. 10.2 mRNA delivery and expression in the tdTomato mice To evaluate mRNA expression in different cell types, CCl 4 -induced liver fibrotic LoxP-flanked tdTomato reporter mice were utilized. Total liver cells (including parenchymal and non-parenchymal cells) were harvested 48 h after i.v. administration of mCre LNP formulations (1.5 mg/kg). Parenchymal cells, specifically primary hepatocytes were harvested by perfusion method as described above. Non-parenchymal cells (including endothelials, HSCs, leucoyctes) were harvested in the supernatant. Cell types were distinguished by size and specific markers by flow cytometry (Hepatocytes: low SSC/HSC, CD45 − ; Endothelial: CD45 − , CD31 + ; Leucocytes, CD45 + ; other cells (mainly HSCs). The percentage of tdTomato + cells in each cell populations were measured and quantified. 10.3 Pharmacokinetics assay CCl 4 -induced liver fibrosis models in C57BL/6 mice were developed as previously described. The mice were i.v. treated with ATRA LNPs containing mRLN, mRLN-PLGF 1 , mRLN-PLGF 3 and mRLN-Fc at a concentration of 1.5 mg/kg. Subsequently, mice were euthanized at 0.25, 1, 3 and 6 days post injection, and both liver tissue and blood samples were harvested. The RLN content in the samples was determined using an ELISA kit (Boster bio) following the manufacturer’s protocol. 11. Statistics Data are expressed as the mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t -test when only two value sets were compared or by ANOVA for comparison between multiple groups via GraphPad Prism 8.02. Exact P values are documented in the figures or figure legends. Difference was considered to be significant if P < 0.05, (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 unless otherwise indicated). Declarations CONFLICTS OF INTERESTS L. M, X.Z.S., Z.Q.Z have filed a patent for the development of the described FORT strategy. AUTHOR CONTRIBUTIONS L.M., X.Z.S., and Z.Q.Z. are responsible for all phases of the research. X.Z.S., Z.Q.Z., P.P.L., B.Y.L., P.X.Q., Y.Z.X., Z.H.Z., C.L.W. and B.M. performed experiments. Y.B.K. and H.Q.J. helped with SANS experiment. W.L. provided purified fusion proteins and cell lysates. W.Z.L. helped with cell uptake and confocal analysis. Q.W. participated in SPR and MST assay. J.Y. and Y.X.L. performed the molecular modeling and dynamics simulation of fusion proteins. Y.F.G. and L.S. performed the lipid membrane simulation. X.Z.S, Z.Q.Z. and L.M. wrote the manuscript. C.L., Y.L.Z., X.G.L., J.Q.L., X.D.X., D.D. and L.M. provided conceptual advice and supervised the study. All the authors discussed the results and assisted in the preparation of the manuscript. ACKNOWLEDGEMENT This research was financially supported by National Key Research and Development Program of China (2023YFC3405000 to L.M), Beijing Natural Science Foundation (Z220022, to L.M), Beijing Municipal Science & Technology Commission (Z231100007223012 to L.M), the National Natural Science Foundation of China (NSFC) grants (HY2021-8, 82373807 to L. M., 82070460, 82270479, HY2021-1 to X.D.X). We thank the State Key Laboratory of Natural and Biomimetic Drugs, Peking University Biological Imaging Facility for confocal, animal, and tissue imaging services. The molecular modelling and dynamics simulations were carried out using the high-performance Linux cluster at the Computing and Data Science Core in the Chinese Institute for Brain Research, Beijing. We thank Keymed Biosciences for providing related proteins. References Huby T, Gautier EL (2022) Immune cell-mediated features of non-alcoholic steatohepatitis. 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Supplementary Files livercrf26fps25.mp4 Supplementary video 1 hsccrf26fps25.mp4 Supplementary video 2 RLNPIGF1100nsout.mp4 Supplementary video 3 RNLPIG3200nsout.mp4 Supplementary video 4 RLNFc200nsout.mp4 Supplementary video 5 SupplementsforNN.docx Cite Share Download PDF Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3746897","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":264364796,"identity":"47e02802-5f34-4016-9b4b-4c88db69f362","order_by":0,"name":"Lei 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Lin","suffix":""},{"id":264364816,"identity":"455c91ab-4ae7-43ae-a477-4b906c662161","order_by":20,"name":"Li Shu","email":"","orcid":"","institution":"Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Shu","suffix":""},{"id":264364817,"identity":"c7539f10-a014-4eed-8267-35a1a75ab945","order_by":21,"name":"Yin Jie","email":"","orcid":"","institution":"Chinese Institute for Brain Research","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Jie","suffix":""},{"id":264364818,"identity":"19b792c7-0e02-4b87-849a-9466753f132d","order_by":22,"name":"Xunde Xian","email":"","orcid":"","institution":"School of Basic Medical Sciences, Peking University","correspondingAuthor":false,"prefix":"","firstName":"Xunde","middleName":"","lastName":"Xian","suffix":""},{"id":264364819,"identity":"622ab421-ee61-4e77-880b-ffaca5d73d5e","order_by":23,"name":"Derfogail Delcassian","email":"","orcid":"","institution":"Department of Bioengineering, UC Berkeley","correspondingAuthor":false,"prefix":"","firstName":"Derfogail","middleName":"","lastName":"Delcassian","suffix":""},{"id":264364820,"identity":"0a730007-2a51-4c24-bf83-dfbacc84241e","order_by":24,"name":"Yifan Ge","email":"","orcid":"","institution":"Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Ge","suffix":""}],"badges":[],"createdAt":"2023-12-13 06:41:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3746897/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3746897/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-51571-8","type":"published","date":"2024-08-27T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50347323,"identity":"b099f026-6ac3-4cb6-a433-ce4fb34e6f16","added_by":"auto","created_at":"2024-01-30 06:49:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1745737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of Fibrosis Overexpression and Retention (FORT) strategy for NASH treatment.\u003c/strong\u003e CBD screened from the endogenous proteins was added to the C terminus of RLN, and coded by mRNA. The therapeutic recombinant mRNAs were encapsulated with a fibrosis-targeted LNP and intravenously (\u003cem\u003ei.v.\u003c/em\u003e) administrated for enhanced mRNA expression and fibrotic liver retention. To achieve enhanced mRNA expression, we fabricated a five component LNPs by substituting 25 mol% of cholesterol in ALC-0315 LNPs with a carboxylic retinoid, ATRA, which shows over a ~10- fold increase in mRNA expression compared to traditional ALC-0315 LNPs in fibrotic and NASH models. Mechanistically, the carboxylic retinoid rearranges on LNPs surfaces during encapsulating mRNA, improving both endocytosis and endosomal release. The added CBD to the therapeutic proteins further extended liver retention. Thereby, the FORT strategy allows the fusion protein to be expressed and anchored \u003cem\u003ein situ\u003c/em\u003e, creating a depot that enhances the anti-fibrotic response. This schematic was created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/6079426e6e9838d9c0356251.png"},{"id":50347912,"identity":"ae5d12ab-3bf1-4c7d-b03d-9c7a5f1a1cef","added_by":"auto","created_at":"2024-01-30 06:57:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3206730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncorporation of carboxylic retinoids in LNPs increased mRNA expression in HSCs and fibrotic livers. \u003c/strong\u003e(a) to (c) Comparison of mRNA expression delivered by representative LNP formulations in wild type (WT) livers and CCl\u003csub\u003e4\u003c/sub\u003e-treated fibrotic livers 6 h after\u003cem\u003e i.v.\u003c/em\u003e injection. mLuc was used as reporter mRNA, 0.25 mg/kg in LNP formulations were administered, n=3 in each group. (d) Structures of different groups of retinoid derivatives incorporated into original LNP formulation as a partial substitution of cholesterol. (e) \u003cem\u003eIn vitro \u003c/em\u003eexpression of mRNA delivered by RD-LNPs formulations in LX-2 cells, primary mouse hepatocytes and lipid-overload primary hepatocytes. Twenty-four hours after incubation of 70 ng mRNA LNPs, the expression was measured using BioTek plate reader. Expression fold changes over ALC-0315 formulations were calculated and presented (n=5). (f) Schematic illustration of CCl\u003csub\u003e4\u003c/sub\u003e induced liver fibrosis mice model. (g) Expression of mLuc 24 h after\u003cem\u003e i.v.\u003c/em\u003e injection of the representative RD-LNPs and the original ALC-0315 LNPs (0.25 mg/kg, n=3 in each group). Quantification of the expression were presented on the right. (h) and (i) The mRNA expression kinetics delivered by the original ALC-0315 formulation and 25% ATRA formulations were compared in CCl\u003csub\u003e4\u003c/sub\u003e-treated (6 week) fibrosis mice. Expression was measured by IVIS at 2, 6, 12, and 24 h after injection. Quantification was presented on the right (0.25 mg/kg, n=3). (j) comparison of expression between the original ALC-0315 and ATRA formulations in hamster NASH models at a dose of 0.25 mg/kg. The expression was measured at 6 h after injection, n=3 in each group, one representative animal from each group were listed. * P \u0026lt; 0.05, **, P \u0026lt; 0.01, n.s., P \u0026gt;0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure220231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/49fc4b6e77f3f04997302fa6.png"},{"id":50347324,"identity":"c8170fc8-617a-40b7-b2df-ce7dd19477a9","added_by":"auto","created_at":"2024-01-30 06:49:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2355238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic investigation of the improved mRNA expression of retinoic acid LNPs. \u003c/strong\u003e(a) Schematic illustration shows the delivery of mCre activates tdTomato expression in Ai14 mice via Cre-mediated genetic deletion of stop cassette. The mice were treated with CCl\u003csub\u003e4\u003c/sub\u003e for 4 weeks before dosing with LNPs. (b) Representative FACS analysis of tdTomato\u003csup\u003e+\u003c/sup\u003e cells in different cell groups within fibrotic liver of CCl\u003csub\u003e4\u003c/sub\u003e-treated mice. The mice were dosed mCre LNPs 48h prior to sacrifice (n=3). (c) Quantification of FACS data. The percentage of positive cells in each cell group was present (n=3). (d) Schematic representation of IF staining of firefly luciferase (green) and α-SMA (red) 24 h after dosing with mLuc LNPs in liver of CCl\u003csub\u003e4\u003c/sub\u003e-treated mice. (e) Cellular internalization of ALC-0315, retinol and ATRA LNPs with inhibition of various internalization pathways (n=3-6, mean ± SEM). (f) and (g) Dose-response curve for the binding interaction between LNPs and RBP-4 protein. \u003cem\u003eKd\u003c/em\u003e values were presented. (h) Characterization of ATRA distribution within LNPs. SANS data (symbols) and best fit (lines) were shown as scattering intensity as a function of the scattering vector (Q) for mRNA-containing ATRA LNPs in D\u003csub\u003e2\u003c/sub\u003eO of gradient ratio (left). Schematic representation of a core-shell structure of LNP (top right) and the percentage of distribution in inner core and outer shell (bottom right). (i) Representative confocal images show the release of mRNA (Cy5) from LNPs (BODIPY). (j) Heatmap summarized the unsaturation contents in PC lipids from endosome of HCs and HSCs (n=4). (k) Schematic of the reconstituted lipid bilayer platform that mimics the endosome membranes from hepatocytes and HSCs. (l) Lipid bilayer composition % for the mimicked endosomal membrane of HCs and HSCs; (m) The fusion efficiency of the three LNPs with the two lipid bilayers; (n) Representative trace of the Texas red labeled LNPs fusing with the mimicked endosomal membrane of HCs (top) and HSCs (bottom). (o) Fusion time recorded in each treatment groups (p) Schematic illustration shows the hypothesis for increased internalization and endosomal escape of ATRA LNPs. * P \u0026lt; 0.05, **, P \u0026lt; 0.01, ****, P \u0026lt; 0.0001, n.s., P\u0026gt;0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure320231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/2dd333b8fbe44eebda0dbb5b.png"},{"id":50347914,"identity":"6ca987cc-ce36-43cf-9932-b112f8aa2f80","added_by":"auto","created_at":"2024-01-30 06:57:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":851516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRational design of mRNA encoded RLN-CBD fusion proteins and verification of their ECM binding capacity.\u003c/strong\u003e (a) Schematic illustration of the structure of mRNA encoded RLN-CBD fusion protein. The ORF encodes the pro-RLN (B, C, A chain) and the CBD domain connected by a flexible GGGS linker. The mRNA was \u003cem\u003ein-vitro\u003c/em\u003etranscribed with the linearized plasmid as the template. mRNA was then encapsulated into ATRA LNPs, transfected into HSCs and subsequently expressed the pro-RLN-CBD. The pro-RLN was then processed to a mature secretable RLN-CBD protein; (b) Lists of 11 endogeneous CBDs screened in our study. (c) Cryo-EM and particle size characterization of fusion protein coded mRNA formulated with ATRA LNP (RLN-Pep K LNP for representative), scale bar represents 100 nm. (d) Schematic illustration for the ELISA method used to assess the collagen binding capacity of the fusion proteins expressed from mRLN-CBD LNPs. (e) The binding of RLN-CBD fusion proteins to collagen measured by ELISA. Binding capacity was calculated by absorbance at 450 nm deducted the value of binding to BSA and further divided with the concentration of proteins in the supernatant (n=3, mean ± SEM). * P \u0026lt; 0.05, **, P \u0026lt; 0.01, ***, P \u0026lt; 0.001, ****, P \u0026lt; 0.0001, n.s., P \u0026gt;0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure420231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/4744f09af76f01bf2ed44b36.png"},{"id":50347913,"identity":"52ee4165-f3a2-4e91-accd-4ac074fd9724","added_by":"auto","created_at":"2024-01-30 06:57:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1901446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emRLN-PLGF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e formulated in ATRA LNPs showed enhanced retention with preserved bio-activity \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) Schematic representations of the recombinant fusion and control proteins. (b) The predicted molecular models and dynamic simulations of fused and un-modified RLN according to the ColabFold. (c) RLN and fusion proteins levels in the supernatant of HSCs transfected with mRNA formulated in ATRA LNPs, measured by ELISA assay using primary antibodies against mouse RLN (n=4, mean ± SEM). (d) The binding of fusion proteins and un-modified RLN to the ECM proteins were measured by ELISA assay. The values of binding to BSA were served as negative control (n=3, mean ± SEM). (e) Affinity (dissociation constant (\u003cem\u003eKd\u003c/em\u003e) values are shown) of fusion proteins and un-modified RLN against collagen II as measured using SPR. \u003cem\u003eKd \u003c/em\u003evalues which were determined from the fitted curves are shown. (f) Schematic representation of the pharmacokinetics of RLN after \u003cem\u003ei.v.\u003c/em\u003e administration of mRNA encoded proteins delivered by ATRA LNPs. Liver and blood were harvested at multiple time points after mRNA LNP treatment. (g) Kinetics of mRNAs-encoded fused and un-modified RLN in liver and blood. mRNAs were formulated in ATRA LNPs and \u003cem\u003ei.v.\u003c/em\u003e administrated to CCl\u003csub\u003e4\u003c/sub\u003e treated liver-fibrotic mice and sacrificed at pre-set timepoints (1.5mg/kg, n=5). (h) The area under curve of (g). (i) Allocation ratio of expressed RLN-PLGF\u003csub\u003e1\u003c/sub\u003e and RLN-Fc in liver and blood 1 day and 3 days post treatment. (j) and (k) Systemic levels of inflammatory cytokines at different times after LNP treatment. (l) Intracellular cAMP levels in THP-1 cells (left) and RAW264.7 cells (right) 12h after fused and unmodified mRLN LNP treatment (1μg mRNA/mL) (n=3, mean ± SEM). (m) Relative mRNA expression of α-SMA and TGF-β in 3T3 cells 24 h after treatment of mRLN or mRLN-CBD LNPs (1μg mRNA/mL) (n=4, mean ± SEM). (n) and (o) Representative IF staining of α-SMA 48h after fused and unmodified RLN mRNA LNPs treatment (μg mRNA/mL) (n=3). The quantity of mean fluorescence intensity was performed by Image J (n=3, mean ± SEM). (p) Dose-response curve for the binding interaction between RLN or RLN-PLGF\u003csub\u003e1\u003c/sub\u003e and RXFP\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e 3T3 cells with and without the presence of collagen IV (n=3). RLN and RLN-PLGF\u003csub\u003e1\u003c/sub\u003e were harvest from the 293F cells lysate transfected with RLN or RLN-PLGF\u003csub\u003e1\u003c/sub\u003e. * P \u0026lt; 0.05, **, P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001, n.s. P \u0026gt; 0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure520231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/2a484192744b9491b6149f52.png"},{"id":50348258,"identity":"e536aee8-ea82-4add-8cc3-d73cab2306a0","added_by":"auto","created_at":"2024-01-30 07:05:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6766518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-fibrosis effects of mRLN-PLGF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e LNPs in CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced fibrotic or MCD-treated NASH models. \u003c/strong\u003e(a) Schematic representations of the CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis models and treatment schedules for ATRA LNP formulated mRLN or mRLN-CBDs (1.5mg/kg mRNA). (b) Serum ALT and AST from CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrotic mice with all treatment groups after 4 doses treatment (n=5, mean ± SEM). (c) Liver index (liver wight/body weight%) of all treatment groups from CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrotic mice model (n=5, mean ± SEM). (d)-(g) Representative IF staining of α-SMA (d), Masson’s trichrome (e), Sirius red (f) and Masson’s trichrome (bottom) and H\u0026amp;E staining (g) in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrotic mice with all treatment groups after treatment (n=5), scale bar represents 250 μm. (h) Quantification of the coverage% area of α-SMA positive in (d), collagen in (e) and (f) respectively. The quantification was performed in three randomly selected fields per mouse (from n=5 biological independent mice per group, mean ± SEM). (i) Schematic representations of the MCD diet induced NASH mouse model and treatment timeline for ATRA LNP formulated mRLN and mRLN-PLGF\u003csub\u003e1 \u003c/sub\u003e(1.5mg/kg mRNA). (j) Liver index (liver wight/body weight %) of all treatment groups from MCD diet induced NASH models (n=5, mean ± SEM). (k) Serum ALT and AST of all treatment groups from MCD mice (n=5, mean ± SEM). (l)-(o) Representative histochemical staining of Sirius red (l), Masson’s trichrome (m), H\u0026amp;E (n) and Oil Red O (o) in MCD mice with all treatment groups (n=5), scale bar represents 250 μm. (p) Quantification of the coverage% area of collagen in (l) and (m) and lipid accumulation area in (o). The quantification was performed in three randomly selected fields per mouse (n=5, mean ± SEM). * P \u0026lt; 0.05, **, P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001, n.s. P \u0026gt; 0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure620231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/023a1e4f6315474ee64a048a.png"},{"id":50347327,"identity":"ca1fc89e-1ce9-41ef-ace5-fb5c90ba20d1","added_by":"auto","created_at":"2024-01-30 06:49:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4710031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow-dose combination of mRLN-PLGF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and mIL-10-PLGF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e delivered by ATRA LNPs for treating hamsters with NASH. \u003c/strong\u003e(a) Schematic representations of the CDHFD induced NASH hamster models and the treatment schedules for single and combinatorial administrations of ATRA LNP formulated mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e and mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e through jugular vein. Hamsters on CDHFD with sham surgery in the jugular vein were served as a control. (b) The schematic structures of RLN-PLGF\u003csub\u003e1\u003c/sub\u003e and IL-10-PLGF\u003csub\u003e1\u003c/sub\u003e recombinant proteins are presented on the right. (c) Representative \u003cem\u003ein vivo \u003c/em\u003eultrasound imaging of livers from the CDHFD induced NASH hamsters from all treatment groups at 0, 6, 12 and 18 d post the first dose treatment. Higher intensity and heterogeneity of the hepatic echogenicity reflected the more aggressive fibrosis conditions. (d), (e) Serum lipid level (TG and TC) in (d), serum ALT and AST in (e) from CDHFD induced NASH hamsters in all treatment groups at 0, 6, 12, 18 d post the first dose treatment. (n=5, mean ± SEM). (f)-(i) Representative histochemical stains for all the treatment groups from CDHFD induced NASH hamsters, including Masson’s trichrome (f), Sirius red (g), Oil Red O (h) and H\u0026amp;E (i) staining (n=5 biological independent hamsters per group), in (f)-(h), scale bar represents 100 μm, in (i), scale bar represents 20 μm; (j) Quantification of the coverage% area of collagen in (f), (g) and fat accumulation area in (h). The quantification was performed in three randomly selected fields per hamster (n=5 biological independent hamsters per group, n=3 fields per hamster, mean ± SEM); (k) lipid level (TG and TC) in liver after treatment; (l) Relative mRNA expression of biomarkers for fibrogenesis, fibrodegradation, lipid-β-oxidation and lipid de novo synthesis in liver of CDHFD induced NASH hamsters with all treatment groups after treatment (n=5, mean ± SEM). * P \u0026lt; 0.05, **, P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001, n.s. P \u0026gt; 0.05. All the schematic illustrations were created with BioRender.com.\u003c/p\u003e","description":"","filename":"figure720231212.png","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/9d56bd80624b3df76d902c24.png"},{"id":63439282,"identity":"15e116cd-f672-4dce-a08f-3292f7ea0c61","added_by":"auto","created_at":"2024-08-28 07:05:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23815694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/78fe56fe-cee9-4b4c-b2ec-9dfb49476651.pdf"},{"id":50347340,"identity":"ffc51d55-cc07-403a-8593-664f61b17f02","added_by":"auto","created_at":"2024-01-30 06:49:05","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":100264335,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 1\u003c/p\u003e","description":"","filename":"livercrf26fps25.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/12a4f2b393c28e96aec7e19e.mp4"},{"id":50347334,"identity":"eceb02b3-9cdb-4f22-b1f2-d46c452fc35b","added_by":"auto","created_at":"2024-01-30 06:49:04","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29663729,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary video 2\u003c/p\u003e","description":"","filename":"hsccrf26fps25.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/ebeedc99c775d2f571d39b33.mp4"},{"id":50347331,"identity":"da53a2c7-2c5e-4bc8-a80e-5515dd758041","added_by":"auto","created_at":"2024-01-30 06:49:03","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9661073,"visible":true,"origin":"","legend":"Supplementary video 3","description":"","filename":"RLNPIGF1100nsout.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/2b0d783742687235ed21ad56.mp4"},{"id":50347332,"identity":"bc5f33cb-f55d-403e-bf8c-894efabc35ff","added_by":"auto","created_at":"2024-01-30 06:49:04","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":41250792,"visible":true,"origin":"","legend":"Supplementary video 4","description":"","filename":"RNLPIG3200nsout.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/79a59a4f255c7968a1a2e35e.mp4"},{"id":50347917,"identity":"40b79cc7-68bf-40d5-a34b-ea48c9fddcae","added_by":"auto","created_at":"2024-01-30 06:57:04","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":52245346,"visible":true,"origin":"","legend":"Supplementary video 5","description":"","filename":"RLNFc200nsout.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/37798479fe873da4d4206554.mp4"},{"id":50347335,"identity":"5bd3da36-f749-436d-b7a4-50809d67f414","added_by":"auto","created_at":"2024-01-30 06:49:04","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12226843,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementsforNN.docx","url":"https://assets-eu.researchsquare.com/files/rs-3746897/v1/87b73e8e30a9ea58aca7c3ef.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"RNA Nanotherapeutics with Fibrosis Overexpression and Retention (FORT) for NASH Treatment","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-alcoholic steatohepatitis (NASH) is a chronic liver inflammation that can progress to fibrosis and cancer, affecting approximately 5% of the population, causing significant mortality worldwide.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Currently, there are limited therapies for the treatment of NASH due to challenges with delivering to, and treating, the fibrotic microenvironment in the liver.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Inspired by the success of glucagon-like peptide 1 (GLP-1) in diabetes treating, protein-based therapies hold promise for NASH treatment.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In animal models of chronic liver injury, the protein relaxin (RLN) has shown benefits.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e RLN interacts with the Relaxin Family Peptide Receptor 1 (RXFP\u003csub\u003e1\u003c/sub\u003e) and inhibits the TGF-β pathway, which is implicated in fibrosis.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Recombinant RLNs are currently undergoing clinical trials for treating heart fibrosis.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Other proteins, such as anti-inflammatory cytokines like IL-10, have also been reported to protect mice from liver fibrosis by counterbalancing hyperactive immune responses.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, these protein therapies are challenging to translate to the clinics due to limited stability, rapid clearance, and lack of organ targeting specificity.\u003c/p\u003e \u003cp\u003eStrategies to extend protein circulation half-life have been successful for some clinically relevant therapeutic proteins (i.e., IL-2, GLP-1 and etc). Approaches have included protein modifications to introduce the Fc fragment of IgG\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, PEGylation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and the addition of long chain alkanes.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Yet, this may not be sufficient to enhance drug concentration at the lesion site and could potentially increase systemic toxicity. In parallel, the design of fusion proteins with specific binding domains has been shown to help anchor protein therapeutics in the lesions.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Unfortunately, the administration of these fusion proteins is limited to local injections in easily accessible disease models, and their manufacturing can be complex and requires further optimization. Therefore, developing strategies for targeted delivery of therapeutic anti-fibrosis proteins and prolonging their retention in fibrotic lesions are critical for NASH treatment.\u003c/p\u003e \u003cp\u003eLipid nanoparticle (LNP) mRNA therapies have shown enormous promise for expression of nucleic acids encoding for target therapeutic proteins.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e For instance, the RNA drug Onpattro is clinically approved for treating transthyretin mediated amyloidosis and highlights the therapeutic potential of LNPs for liver diseases. LNPs often preferentially deliver nucleic acid cargo to the liver. This is thought to be due, in part, to adsorption of ApoE proteins to the LNP surfaces, and targeting other organs through chemical modifications to LNPs is an expanding research area.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough the liver is considered a relatively accessible organ for mRNA LNPs in healthy patients, it becomes less amenable to therapeutic intervention during fibrosis. Hepatic stellate cells (HSCs), residing between liver sinusoidal endothelial cells (LSECs) and hepatocytes, become activated during fibrogensis.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e These activated HSCs (aHSCs) deposit excessive amount of extracellular matrix (ECM) proteins in fibrotic lesions. This exacerbates inflammation, contributes to capillarization and closure of fenestrae, thus restricting drug delivery to the liver.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The NASH microenvironment is therefore exceptionally challenging for therapeutic delivery of mRNA LNPs.\u003c/p\u003e \u003cp\u003eHere, we present a new strategy for delivering therapeutic proteins to hepatic NASH lesions via mRNA LNPs in a process we term \u0026ldquo;Fibrosis overexpression and retention (FORT)\u0026rdquo;. Briefly, we designed a new LNP which incorporates retinoid derivative ligands to facilitate targeted delivery to HSCs. Retinoid derivatives have previously been shown to facilitate fibrotic accumulation of therapeutic regimens due to \u0026ldquo;attach\u0026rdquo; to retinol-binding protein-4 (RBP-4). Among the analogues tested, carboxylic retinoids, not conventional alcoholic or ester-derived retinoids have shown improved mRNA expression in fibrotic livers. Mechanistic investigations revealed that the carboxylic retinoids rearranged on the outer shell of LNPs during mRNA encapsulation, resulting in improved binding to RBP-4, enhanced internalization, sprouting, and endosomal escape. We use these LNPs to deliver mRNA encoding for engineered proteins which co-express an endogenous peptide domain to \u0026ldquo;anchor\u0026rdquo; proteins into the fibrotic ECM. Using this approach, our lead ligand candidate (all-trans retinoic acid, ATRA) demonstrated a\u0026thinsp;~\u0026thinsp;10- fold increase of mRNA expression in fibrotic livers compared to commercial ALC-0315 formulations. We used this LNP to deliver mRNA encoding for an anti-fibrotic RLN protein fused to an optimized collagen binding domain (CBD) from placenta growth factor (PLGF). We demonstrate 80% retention of the fusion protein in the liver. We further demonstrate therapeutic reduction in fibrosis in three clinically relevant NASH models. We believe the FORT approach could be widely applicable to the delivery and anchoring of therapeutic proteins to less accessible fibrotic microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInclusion of retinoic acid but not retinol improves mRNA delivery to fibrotic livers\u003c/h2\u003e \u003cp\u003eWe hypothesized that LNP-mediated mRNA expression in the liver would be reduced in models of chronic liver inflammation.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). mLuc LNPs were administered via intravenous (\u003cem\u003ei.v.\u003c/em\u003e) injection to an experimental late-stage liver fibrosis model induced by 6-week administration of tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) and a NASH hamster model induced by 10-week choline deficient high fat diet (CDHFD) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2\u003c/b\u003e). We observed a 6\u0026thinsp;~\u0026thinsp;20- fold decrease in mLuc expression delivered by all three types of LNPs in fibrotic models compared to WT controls.\u003c/p\u003e \u003cp\u003eNext, 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 strategy\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, 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.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eIn vivo\u003c/em\u003e, retinol and selected retinol metabolites bind to the serum protein RBP-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), which then facilitates cellular endocytosis in HSCs.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). 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).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e 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 \u003cb\u003e(Table. S1)\u003c/b\u003e. Most of the first-generation retinoids exhibited high binding affinity (\u003cem\u003eKd\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026thinsp;~\u0026thinsp;5\u0026micro;M), consistent with previous report,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e while the second generation showed moderate binding (\u003cem\u003eKd\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30\u0026thinsp;~\u0026thinsp;60 \u0026micro;M).\u003c/p\u003e \u003cp\u003eBased 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Retinol derivatives were included at 5 mol% to 25 mol% within the cholesterol component, other original lipid ratio was maintained (\u003cb\u003eTable S2\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee indicate that incorporating carboxylic acid retinol derivatives, as opposed to alcohol or ester derivatives, significantly enhanced mRNA delivery in aHSCs (\u003cb\u003eFig. S3\u003c/b\u003e). 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 (~\u0026thinsp;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).\u003c/p\u003e \u003cp\u003eTo validate these trends \u003cem\u003ein vivo\u003c/em\u003e, RD-LNPs containing ATRA, bexarotene, A-VA and retinol (all at a 25 mol% replacement of cholesterol, \u003cb\u003eTable S2\u003c/b\u003e) 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 (\u003cb\u003eFig. S4, Table S2\u003c/b\u003e). We injected these particles into mice that were pre-treated with CCl\u003csub\u003e4\u003c/sub\u003e 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 ~\u0026thinsp;8.0-, 3.7- and 2.8- fold increase in luciferase expression compared to the original ALC-0315 formulation in fibrotic livers, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g, \u003cb\u003eFig. S5\u003c/b\u003e). This observation aligned with the \u003cem\u003ein vitro\u003c/em\u003e transfection study conducted in LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), highlighting the potential of targeting aHSCs for LNP delivery in fibrotic livers.\u003c/p\u003e \u003cp\u003e 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 CCl\u003csub\u003e4\u003c/sub\u003e for 6 weeks, ATRA LNPs enhanced protein expression\u0026thinsp;~\u0026thinsp;10- fold compared to the ALC-0315 formulations, however this increased expression was not observed in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Over 95% of mRNA was expressed in liver rather than other organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, \u003cb\u003eFig. S6\u003c/b\u003e). 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 (\u003cb\u003eFig. S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFinally, 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 CCl\u003csub\u003e4\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, \u003cb\u003eFig. S8\u003c/b\u003e). Together, these data demonstrate improved LNP delivery and mRNA translation to fibrotic liver \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e through incorporation of carboxylic retinoids in the LNP formulations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRearrangement of retinoic acid in LNP facilitates endocytosis and endosomal release of RNA in aHSCs\u003c/h2\u003e \u003cp\u003eTo validate the role of aHSCs in mediating enhanced mRNA expression in fibrotic livers treated with RD-LNPs, we looked protein expression in CCl\u003csub\u003e4\u003c/sub\u003e treated tdTomato reporter mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). 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\u003csup\u003e+\u003c/sup\u003e cells were observed in HSC-like population treated with ATRA LNPs compared to those treated with ALC-0315 LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cb\u003eFig. S9\u003c/b\u003e). This result aligns with the \u003cem\u003ein vitro\u003c/em\u003e study and suggests that ATRA LNPs tend to accumulate in the fibrotic area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). 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\u003csup\u003e+\u003c/sup\u003e fibrotic area in mice treated with ATRA LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eWe 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\u003cb\u003e)\u003c/b\u003e 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.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e 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 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, \u003cb\u003eFig. S10)\u003c/b\u003e. Specifically, ATRA and A-VA LNPs showed 1.5\u0026thinsp;~\u0026thinsp;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 (\u003cb\u003eFig. S12\u003c/b\u003e). Macropinocytosis was identified as a major pathway for LNP uptake for all LNPs tested, consistent with previous reports.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In a separate study, we also knocked down the RBP-4 receptor STRA6 with siRNA 24 h prior to adding LNPs (\u003cb\u003eFig. S11\u003c/b\u003e) in aHSCs. Interestingly, knockdown of STRA6 significantly reduced the uptake efficiency of retinoic acid LNPs (ATRA and A-VA LNPs) by approximately 2\u0026thinsp;~\u0026thinsp;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 (\u003cb\u003eFig. S12\u003c/b\u003e). 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 (\u003cb\u003eFig. S10\u003c/b\u003e). 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 (\u003cb\u003eFig. S13\u003c/b\u003e); however, the addition of retinoids improved LNP binding to RBP-4. Interestingly, encapsulating mRNA into retinol LNPs didn\u0026rsquo;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef \u003cb\u003eand g\u003c/b\u003e). 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\u0026ndash;5 (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep). To gain further insights into the LNP structure, we employed Small-Angle Neutron Scattering (SANS) as previously described.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The distribution of the ATRA within LNPs was elucidated by varying the content of deuterated water (D\u003csub\u003e2\u003c/sub\u003eO) to match the scattering length densities of different region of the particle (\u003cb\u003eTable S9\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh)\u003c/p\u003e \u003cp\u003eAdding 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 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, \u003cb\u003eFig. S14, S15\u003c/b\u003e). The result of lipidomics revealed that hepatocyte endosome has a lower saturation lipid content compared to HSCs\u0026rsquo;, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek).\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The ratio of DOPC and DPPC was tuned to match the saturation levels of the endosomal membranes from HSCs and hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el). Membrane rigidity was confirmed via measuring the diffusion coefficient using fluorescent correlation spectroscopy (\u003cb\u003eFig. S16\u003c/b\u003e). 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 (\u0026gt;\u0026thinsp;90% fused) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em, n, \u003cb\u003eVideo S1\u003c/b\u003e). However, in the rigid membrane mimicking HSC endosomes, almost no fusion events were observed for ALC-0315 LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em, n, \u003cb\u003eVideo S2\u003c/b\u003e). Nevertheless, ATRA LNPs exhibited over 50% fusion at the end point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em), with faster diffusion compared to other LNP treated membranes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo). 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 (\u003cb\u003eFig. S16\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep). Overall, these findings enhance our understanding of the mechanisms underlying the improved transfection efficiency observed with retinoic acid LNPs targeting HSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003emRNA encoding collagen binding recombinant proteins improved retention in fibrotic region\u003c/h2\u003e \u003cp\u003eFollowing 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\u0026rsquo;s local expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Through RNA sequencing of WT hamsters and hamsters with NASH, we identified \u003cem\u003ecol1α1\u003c/em\u003e and \u003cem\u003ecol1α2\u003c/em\u003e as major ECM proteins significantly increased in NASH livers \u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e(\u003c/span\u003e\u003cb\u003eFig\u003c/b\u003e. \u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eS17)\u003c/span\u003e.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e 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 \u003cem\u003ein vivo\u003c/em\u003e 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.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Therefore, we retained the original mRNA sequence encoding the pro-RLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). As the B-chain\u0026rsquo;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.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e 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 \u003cem\u003ein vitro\u003c/em\u003e transcription (\u003cb\u003eFig. S18\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). 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 (\u003cb\u003eFig. S19\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The results demonstrated that the addition of CBD Pep K to RLN led to strong and versatile binding to ECM proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and S20).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Pep K is derived from collagen binding domains found in placenta growth factor-2 (PLGF-2\u003csub\u003e123\u0026minus;144\u003c/sub\u003e), and was selected as the CBD domain candidate for the remainder of our study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRLN-PLGF\u003csub\u003e1\u003c/sub\u003e mRNA delivered by ATRA LNPs showed improved retention and comparable activity in fibrotic livers\u003c/h2\u003e \u003cp\u003eTo 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-PLGF\u003csub\u003e1\u003c/sub\u003e and RLN-PLGF\u003csub\u003e3\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. 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-PLGF\u003csub\u003e1\u003c/sub\u003e and RLN-Fc outside the receptor binding domain of RLN, while RLN-PLGF\u003csub\u003e3\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, \u003cb\u003eVideo S3-S5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003emRNA encoding for the four proteins was encapsulated (separately) in ATRA LNPs and delivered to HSCs (\u003cb\u003eFig. S21\u003c/b\u003e). Interestingly, results revealed that although the expression of RLN-PLGF\u003csub\u003e3\u003c/sub\u003e could be detected through IF staining, the level of protein secretion was significantly lower compared to the other fusion proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec 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.\u003c/p\u003e \u003cp\u003eTo assess the collagen binding capabilities, we performed the sandwich ELISA assay, which showed that RLN-PLGF\u003csub\u003e1\u003c/sub\u003e demonstrated improved binding to collagen compared to RLN-PLGF\u003csub\u003e3\u003c/sub\u003e, RLN and RLN-Fc. We then purified RLN, RLN-PLGF\u003csub\u003e1\u003c/sub\u003e and RLN-Fc, and determined their \u003cem\u003eKd\u003c/em\u003e values against collagen II using surface plasmon resonance (SPR). Results showed a relatively high binding affinity with ~\u0026thinsp;20 nM of \u003cem\u003eKd\u003c/em\u003e value for the RLN-PLGF\u003csub\u003e1\u003c/sub\u003e fusion protein. In contrast, unmodified RLN and RLN-Fc did not exhibit significant binding to collagen II (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo explore the protein retention \u003cem\u003ein vivo\u003c/em\u003e, we delivered 1.5 mg/kg of each of the four mRNAs to CCl\u003csub\u003e4\u003c/sub\u003e-treated mice using ATRA LNPs (1.5 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). RLN-PLGF\u003csub\u003e1\u003c/sub\u003e, but not RLN-PLGF\u003csub\u003e3\u003c/sub\u003e or RLN-Fc, improved the accumulation of RLN within the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg \u003cb\u003eand h\u003c/b\u003e). The AUC of RLN in mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e group was approximately 2 times higher than mRLN-Fc and 3 times higher than unmodified RLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg \u003cb\u003eand h\u003c/b\u003e). Notably, RLN-Fc exhibited prolonged systemic circulation, while minimal RLN-PLGF\u003csub\u003e1\u003c/sub\u003e was detected in the blood during sample collection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg \u003cb\u003eand h\u003c/b\u003e). Three days post injection, over 80% of RLN-PLGF\u003csub\u003e1\u003c/sub\u003e was retained in the liver, whereas approximately 80% of RLN-Fc were circulated in the bloodstream (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). 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-PLGF\u003csub\u003e1,\u003c/sub\u003e confirming systemic toxicity was reduced using the RLN-PLGF\u003csub\u003e1\u003c/sub\u003e modality (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej, k \u003cb\u003eand Fig. S23\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe biological activities of the recombinant proteins were measured using a cAMP activation assay. All mRNA encoding fusion proteins activated cAMP, with RLN-PLGF\u003csub\u003e3\u003c/sub\u003e showing slightly lower activation compared to other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el). 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-PLGF\u003csub\u003e3\u003c/sub\u003e 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-PLGF\u003csub\u003e3\u003c/sub\u003e would have compromised the biological activities due to intramolecular interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em-o).\u003c/p\u003e \u003cp\u003eRLN acts through the RXFP\u003csub\u003e1\u003c/sub\u003e receptor.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e MST assays were performed to examine if RLN to RXFP\u003csub\u003e1\u003c/sub\u003e binding were negatively impacted using the collagen anchored RLN-PLGF\u003csub\u003e1\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ep and S24). Cell lysates from 293F cells containing free RLN-His-tag protein and RLN-PLGF\u003csub\u003e1\u003c/sub\u003e-His-tag protein were incubated with collagen IV at the saturation concentration. RXFP\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e/RXFP\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e cells were then incubated with free and collagen treated RLN-His-tag protein and RLN-PLGF\u003csub\u003e1\u003c/sub\u003e-His-tag protein. The addition of collagen did not significantly affect the binding of RLN-PLGF\u003csub\u003e1\u003c/sub\u003e to RXFP\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e cells. No significant binding was observed for RXFP\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (\u003cb\u003eFig. S24\u003c/b\u003e). Collectively, these findings demonstrate that the fusion of RLN with 1 unit of the PLGF domain facilitates collagen anchoring whilst maintaining RLN function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003emRLN-PLGF\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e \u003cb\u003ein ATRA LNPs reduces fibrosis and fatty liver in a CCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-treated fibrosis and MCD models\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe assessed the anti-fibrosis effect of the FORT strategy in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis mouse models (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Four doses of ATRA LNPs containing different mRNA constructs (encoding RLN, RLN-PLGF\u003csub\u003e1\u003c/sub\u003e, RLN-PLGF\u003csub\u003e3\u003c/sub\u003e and RLN-Fc) or empty LNPs were \u003cem\u003ei.v.\u003c/em\u003e administrated to CCl\u003csub\u003e4\u003c/sub\u003e-treated mouse. As a positive control, we also orally administered obeticholic acid (OCA), a clinically investigated small molecule for treating NASH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e OCA reduced liver index and collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-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.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eATRA 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 \u003cb\u003e(Fig. S25)\u003c/b\u003e. Notably, treatment with mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e LNPs demonstrated the most significant benefits in reversing liver fibrosis. It led to a\u0026thinsp;~\u0026thinsp;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\u0026rsquo;s trichrome and Sirius red staining, more pronounced than the positive control OCA group. H\u0026amp;E staining also revealed reduced liver damage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-f\u003cb\u003e)\u003c/b\u003e. In contrast, mRLN-PLGF\u003csub\u003e3\u003c/sub\u003e LNPs failed to show significant improvement of liver damage which supports our earlier in vitro data (\u003cb\u003eFig. S25\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e. As in the CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis, mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e demonstrated enhanced therapeutic effects compared to the unmodified mRLN LNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej-p\u003cb\u003e)\u003c/b\u003e. In fact, the reduction in collagen coverage in the liver was 2- fold greater in the mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e group than in mRLN group, as evidenced by Sirius red and Masson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el \u003cb\u003eand m)\u003c/b\u003e. In the mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e group, significantly lower levels of liver index, AST/ALT levels were observed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej \u003cb\u003eand k)\u003c/b\u003e. Interestingly, Oil red O staining revealed that mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e 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 (~\u0026thinsp;2-10- fold) in inflammatory cytokines (e.g. IL-1β, IL-6) productions in mice treated with mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e LNPs group compared to mRLN LNPs (\u003cb\u003eFig. S26\u003c/b\u003e). Additionally, there was a slight decrease in expression of fatty acid uptake genes (\u003cem\u003eFabp1, Cd36, Lipin1\u003c/em\u003e) and lipogenesis genes (\u003cem\u003eSrebp1c, Fasn, Dgat2\u003c/em\u003e), along with upregulation of genes involved in lipid oxidation \u003cem\u003e(Acot1\u003c/em\u003e). These findings suggest that ATRA LNPs formulated mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e have the potential to remodel the NASH microenvironment by affecting both fibrotic and lipid biosynthesis pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLow-dose combination of mRLN-PLGF\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand mIL-10-PLGF\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eLNP leads to outstanding performance in hamster models with NASH\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cb\u003eFig. S2\u003c/b\u003e). In addition to mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e, we introduced IL-10-PLGF\u003csub\u003e1\u003c/sub\u003e, another CBD-based fusion protein, by fusing PLGF peptide on the C terminus \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Adding PLGF\u003csub\u003e1\u003c/sub\u003e on IL-10 also improved the binding to ECM proteins (\u003cb\u003eFig. S20\u003c/b\u003e). Both mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e and mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and S27). Remarkably, combination of mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e and mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e LNP substantially ameliorated liver fibrosis and inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-k). This was characterized by the reduced and more homogenous echo intensity, as well as the lower levels of ALT/AST \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e and TC/TG compared to the sham group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed \u003cb\u003eand k)\u003c/b\u003e. Interestingly, mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e LNPs significantly reduced TC/TG levels in the circulation, while mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e 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 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef \u003cb\u003eand g)\u003c/b\u003e. Combo therapy almost eliminated the accumulation of lipid droplets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh \u003cb\u003eand i)\u003c/b\u003e. We further investigated the genes associated with fibrosis and lipid metabolism. mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e LNP monotherapy induced a more substantial reduction of pro-fibrogenic factors, while the combo therapy fell in between, with ~\u0026thinsp;5.7- fold, ~\u0026thinsp;12.8- fold, ~\u0026thinsp;8.0- fold downregulation of \u003cem\u003eTGF-β\u003c/em\u003e, \u003cem\u003eα-SMA\u003c/em\u003e, \u003cem\u003eCOL1α1\u003c/em\u003e, along with significant upregulation of \u003cem\u003eMMPs\u003c/em\u003e, when compared to the sham group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e. mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e showed slight inhibition of lipogenesis genes (\u003cem\u003eSCD1\u003c/em\u003e, \u003cem\u003eSREBP1C\u003c/em\u003e), consistent with those observed in the MCD models. In contrast, mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e treatment led to ~\u0026thinsp;3.0-, ~\u0026thinsp;7.0- and ~\u0026thinsp;3.6- fold decrease of these genes (\u003cb\u003eFig. S27\u003c/b\u003e). The combination of both resulted in decreased lipid synthesis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e. Notably, promotion of fatty acid β-oxidation was observed in both therapies \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e. 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 (\u003cb\u003eFig. S28\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DICUSSIONS","content":"\u003cp\u003eFibrotic diseases are a leading cause of morbidity and mortality worldwide. Here we present a new approach to treating fibrotic diseases, termed \u0026ldquo;Fibrosis overexpression and retention (FORT)\u0026rdquo;. In this two-part strategy, we design 1) retinoic acid LNPs which distributed retinoids on the surface of the particles, facilitating RBP-4 mediated endocytosis and sprouting/fusion-mediated endosome escape in HSCs, and 2) modified mRNAs which encode for CBDs allowing us to anchor expressed therapeutic fusion proteins to fibrotic ECM. We believe this is the first time that fibrotic lesions have been targeted and treated in this way. This approach offers advantages over current modalities as it confines the therapeutic protein to the disease region, extending the duration of action and minimizing systemic exposure.\u003c/p\u003e \u003cp\u003eFibrotic liver diseases such as NASH and cirrhosis are characterized by capillary base thickening and ECM accumulation. These biophysical changes increase difficulty for therapeutic delivery. In our study, we found commercial LNP formulations were ~\u0026thinsp;10- fold less effective at delivering mRNA to NASH animal models. We proposed the physical incorporation of FDA-approved molecules to enhance mRNA delivery to HSCs in fibrotic livers. Retinoid derivatives were chosen due to their high no-observed-adverse-effect level (NOAEL) in patients and their established suitability for targeting HSCs. Although targeted moieties such as antibodies, nanobodies and aptamers have been widely explored to improve HSC targeting, they often present challenges in terms of scaling up complexity and batch-to batch variability.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In contrast, the physical incorporation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e of hydrophobic moieties into lipid bilayers presents a simple targeting approach with ease of manufacturing. This approach also facilitates incorporation of small molecules over a wide dose range.\u003c/p\u003e \u003cp\u003eOur study focused on exploring the structure-activity relationship of the retinoid derivatives by physically encapsulating various moieties within LNPs. Surprisingly, conventional retinol or uncharged esters did not exhibit effective targeting to HSCs, while carboxylic acid derivatives demonstrated improved targeting. Mechanistic studies suggested that these derivatives may induce a spatial reorganization of lipids when condensed with mRNA, affecting RBP-4-mediated cell uptake. Additionally, we discovered that the endosomal membrane of HSCs is more rigid than that of hepatocytes. ATRA LNPs with enhanced sprouting and fusion rate demonstrated superior fusion with HSC-endosome mimicking membranes, potentially promoting RNA release.\u003c/p\u003e \u003cp\u003eTo enhance therapeutic efficacy, new strategies enabling protein retention without compromising biological activity are required. Previous studies have demonstrated that binding therapeutic antibodies or proteins to collagen can enhance local accumulation. However, the applications have been limited by delivery challenges. In our study, we delivered designed mRNA encoding for therapeutic proteins fused to CBD to facilitate retention in the NASH fibrotic lesion. We selected a CBD motif from endogenous protein domains, as opposed to peptide sequences obtained from display techniques, since the endogenous CBDs offer advantages such as lower immunogenicity and higher specificity. Notably, we found that the addition of multiple CBDs does not further enhance binding or efficacy. In contrast, it might induce mis-folding, compromising therapeutic efficacy. Therefore, we chose to combine therapeutic proteins with a single CBD, identified from PLGF.\u003c/p\u003e \u003cp\u003eRLN is a promising therapeutic protein for treatment of fibrosis currently in clinical trials.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e However, clinical failures of RLN peptides have been primarily attributed to undesirable pharmacokinetics.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Additionally, RLN lacks an intrinsic CBD to facilitate its interaction with the ECM. We demonstrate that our modified RLN-PLGF\u003csub\u003e1\u003c/sub\u003e protein improved fibrotic liver accumulation of RLN protein and increased anti-fibrosis activity in three NASH models. We also extended the use of FORT strategy to other secretable proteins such as IL-10.\u003c/p\u003e \u003cp\u003eIn conclusion, we have developed a new type of mRNA LNP therapy for the treatment of fibrosis, termed FORT. By incorporating an FDA-approved retinoic acid in LNPs, the expression of RNA therapeutics in fibrotic liver was significantly improved. Further, the addition of an endogenous CBD domain from PLGF to mRNA sequences generates a fusion protein with improved retention in fibrotic lesions. Delivery of therapeutic proteins using these strategies ameliorate fibrosis in animal models. We believe this approach can be broadly applied to other chronic inflammatory diseases that are not amenable to direct injection but can be targeted using LNPs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAll the ionizable lipids containing DLin-MC3-DMA (MC3), 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) and 1-octylnonyl 8-(2-hydroxyethyl)6-0x0-(undecyloxy)hexyl amino-octanoate (SM-102) were purchased from Avanti Polar Lipids, Inc. Helper lipids containing cholesterol, 1,2-Dioctadecanoyl-sn-glycero-3-phophocholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) were bought from A.V.T. Pharmaceutical Tech Co., Ltd. Small-molecular retinoids containing retinol, 4-keto-retinol, all-trans-retinoic acid (ATRA), 13-cis retinoic acid (13-CRA), Acitretin (A-VA), were purchased from Sigma-Aldrich, 9-cis retinoic acid (9-CRA), fenretinide and acetyl-retinol were purchased from Macklin Co., Ltd, bexarotene and tamibarotene were obtained from MedChemExpress Co., Ltd. Firefly luciferase mRNA (mLuc) was provided by Proxybio. Collagen I, II was purchased from Sino Biological and Collagen IV from Sigma. Human and mouse RBP-4 were purchased from Sino Biological Inc. Purified RLN, RLN-PLGF\u003csub\u003e1\u003c/sub\u003e, RLN-Fc and cell lysates containing RLN and RLN-PLGF\u003csub\u003e1\u003c/sub\u003e were provided by KeyMed Biosciences. The small-molecular uptake inhibitors and carbon tetrachloride were purchased from Macklin Co., Ltd. Methionine-choline-deficient (MCD) diet and choline-deficient high-fat diet (CDHFD, 45% kcal) was purchased from Dytes Inc. All the plasmid sequences were provided by Genscript Co.,Ltd. Primers and siRNA were from Tsingke Biotechnology Co.,Ltd. All cell lines and fetal bovine serum (FBS) were purchase from Procell Life Science \u0026amp; Technology Co.,Ltd. Other reagents for basal culture were bought from Meilunbio Co., Ltd. ELISA kits for mouse IL-6, TNF-α, IL-1β and IFN-γ were purchased from Solarbio Science \u0026amp; Technology Co.,Ltd., mouse RLN-1 from Boster Bio and cAMP from Elabscience Biotechnology Co.,Ltd.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1. mRNA synthesis\u003c/h2\u003e \u003cp\u003emRLN, mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e, mRLN-Fc, mIL-10-PLGF\u003csub\u003e1\u003c/sub\u003e, mLuc and mCre were synthesized using T7 polymerase mediated \u003cem\u003ein vitro\u003c/em\u003e transcription (IVT) system from linearized pUC57 plasmid vectors containing T7 promoter, 5ʹ and 3ʹ untranslated regions (UTRs) and a poly A tail (100 nt). The clean-cap AG 5\u0026rsquo; capping (Cap 1) and 1-methylpsuedo-uridine UTP were added to the transcription reaction. The uridine-5\u0026rsquo;triphosphate (UTP) was fully replaced with 1-methylpsudeo-uridine UTP to improve protein translation and minimize immunogenicity generated from synthesized mRNA. IVT reactions were conducted according to the manufacturers\u0026rsquo; protocols (Hongene Biotech Inc., China). The mRNAs encoding fusion proteins containing 11 different CBD domains (i.e. RLN and IL-10) for initial screening were synthesized from linearized pUC57 plasmid vectors with the absence of poly A sequence. Poly A were added after IVT reactions using Poly A Polymerase Tailing Kit (Beyotime Biotechnology). The CDS sequences of mRNAs used in the current study were listed in \u003cb\u003eTable S8\u003c/b\u003e. Purity of the linearized plasmids and synthesized mRNAs were validated by gel electrophoresis (\u003cb\u003eFig. S20\u003c/b\u003e and \u003cb\u003eS23\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2. Lipid nanoparticle preparation and characterization\u003c/h2\u003e \u003cp\u003eLNPs were prepared either by hand mixing or microfluidic mixing as previously described. Briefly, an aqueous solution of the mRNA and an ethanolic solution of the lipid components were mixed at a ratio of 3:1, respectively. The ethanol phase consists of ionizable ALC-0315 (Avanti), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC, AVT, China), cholesterol (AVT, China) and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000, AVT, China) and a series of FDA approved retinoids at the predetermined molar ratio (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The aqueous phase was prepared in 10 mM citrate buffer containing 0.14 \u0026micro;g/\u0026micro;L mRNA. After mixing, the obtained LNPs were dialyzed against 1\u0026times; PBS in dialysis bag at 4\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003emRNA concentration and encapsulation efficiency of LNP were measured using Quant-it RiboGreen RNA assay (Invitrogen). The hydrodynamic diameter and zeta potential were measured by dynamic light scattering (Zetasizer Nano ZSP, Malvern). The morphology of LNPs were characterized by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). For cryo-EM, the prepared LNP were dialyzed in 20 mM Tris (pH 7.4) containing 8% sucrose 4\u0026deg;C overnight, and then concentrated to 0.5 mg/ml total RNA by ultrafiltration. Cryo-EM image was acquired using Themis 300 (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eTo evaluate the plasma stability of LNPs, ALC-0315, retinol and ATRA LNPs were incubated in DMEM culture medium (pH 7.4) containing 10% FBS for 12 h at 37 ℃. The particle sizes of LNPs were measured by a Zetasizer at predetermined time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3. Small angle neutron scattering (SANS)\u003c/h2\u003e \u003cp\u003eIn this work, SANS was performed on the Small Angle Neutron Scattering (SANS) instrument at China Spallation Neutron Source (CSNS).\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The incident neutrons with wavelength of 1\u0026ndash;10\u0026Aring; were defined by a double-disc bandwidth chopper, which is collimated to the sample by a pair of apertures. The experiment used the sample to detector distance of 4 m and a sample aperture of 6 mm. The 1m square detector array composed of 120 linear He-3 gas tubes with the diameter of 8 mm, which covers the Q-range between 0.01\u0026Aring;-1 and 1\u0026Aring;-1. The presented data correspond to ~\u0026thinsp;120 min of data collection time for each sample (@140kW). For all SANS data, background signals from the solvent, sample cell and the instrument were subtracted by separate runs to measure their scattering contributions. Neutron data were normalized and corrected for transmission and detector efficiency, and set to absolute units. Modeling and simulation details are provided in \u003cb\u003eSupplementary Methods\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4. Microscale thermophoresis (MST) assay\u003c/h2\u003e \u003cp\u003eThe binding affinities between RBP-4 to free retinoids, empty and mRNA loaded LNP were measured using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany). Firstly, RBP-4 was adjusted to a concentration of 10 \u0026micro;M and labeled with MonolithTM NT.115 protein Labeling Kit RED-NHS (Nanotemper Technologies, Germany), following the manufacturer\u0026rsquo;s protocols. The labeled RBP-4 was then purified by gel filtration and diluted to a 250 nM solution using PBS containing 0.05% Tween 20 (PBS-T), ensuring that the fluorescent intensity of RBP-4 during the MST assay was approximately 500 response units (RU). LNPs with an initial concentration of 1 mg/mL (total lipid) were serially diluted (16-point, 1:1 dilution in PBS-T) and mixed in equal volume with the pre-diluted RBP-4. The mixture was incubated for 1h at room temperature. After incubation, the samples were loaded into premium treated capillaries and measured using the NanoTemper Monolith NT.115 instrument. The dissociation equilibrium constant (KD) values were fitted according to the KD Model (1:1 binding mode), by the NanoTemper Monolith affinity software MO. Affinity Analysis v2.3 (NanoTemper Tecchnologies, Germany).\u003c/p\u003e \u003cp\u003eThe binding affinity measurement between the RLN fusion protein (with or without the presence of collagen) and surface receptor RXFP\u003csub\u003e1\u003c/sub\u003e was carried out using crude cell lysates, and measured by NanoTemper Monolith NT.115 instrument following similar protocols as described above. Details are provided in the \u003cb\u003eSupplementary Materials\u003c/b\u003e.\u003c/p\u003e \u003ch2\u003e5. In vitro collagen binding assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates at a density of ~\u0026thinsp;5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells overnight and treated with mRNA-LNP. After 24 h of incubation, the cell culture supernatant was collected and incubated in 96-well ELISA plates that were pre-coated with collagen I, collagen II, collagen IV and BSA (10 \u0026micro;g/mL). The plates were then blocked with a solution of 2% BSA in PBS-T. Following a 1 h incubation at 37\u0026deg;C, the supernatant was removed, and the plates were washed and incubated with rabbit anti-Flag-tag antibody for 1 h at 37\u0026deg;C. After incubation, the plates were washed and incubated with HRP-conjugated goat anti-rabbit antibody for additional 1 h at room temperature. The collagen binding capacity was determined by measuring the absorbance of colorimetric TMB substrate, which reacts with HRP at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e6. Surface plasmon resonance\u003c/h2\u003e \u003cp\u003eThe SPR binding assays were performed using Biacore 8K\u003csup\u003e+\u003c/sup\u003e (Biacore, Cytiva). Recombinant collagen II (Sino Biological) was immobilized onto a CM5 sensor chip using standard amine coupling at 25\u0026deg;C. The reference flow cell was activated and subsequently blocked with BSA. The immobilization levels of collagen II were consistently around 3000 RU. To evaluate the binding affinity, various concentrations of RLN, RLN-PLGF\u003csub\u003e1\u003c/sub\u003e, RLN-Fc (6.25, 12.5, 25, 50, 100 and 200 \u0026micro;M) were injected into the channel. The binding assays involved subtracting the response observed in the reference flow cell containing BSA to account for non-specific binding. Regeneration of the sensor chip was achieved by performing extended washes with NaOH (5 mM) after each sample injection. The dissociation constants (KD) were determined by fitting the obtained data sets to a steady-state affinity model using the Biacore 8K Evaluation Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e7. Molecular modeling and dynamics simulation of fusion proteins\u003c/h2\u003e \u003cp\u003eTo predict the molecular models of all fusion proteins, we employed ColabFold V1.5.2\u003csup\u003e31\u003c/sup\u003e and run it in Google Colaboratory. We used the crystal structure (PDB: 6RLX) of human RLN-2 peptide\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e as a template to predict the RLN-PLGF\u003csub\u003e1\u003c/sub\u003e fusion protein. Among the generated models, the one with the highest pLDDT score (predicted local distance difference test) was selected as the best model for the further modeling and molecular dynamics experiments. For predicting RLN-PLGF\u003csub\u003e3\u003c/sub\u003e and RLN-Fc, the previously predicted RLN-PLGF\u003csub\u003e1\u003c/sub\u003e model and the crystal structure of human RLN-2 peptide (PDB:6RLX) were used as templates. Again, the models with the highest pLDDT score were selected as the best models for the subsequent molecular dynamic experiments.\u003c/p\u003e \u003cp\u003eTo carry out the molecular dynamics simulations, Groningen Machine for Chemical Simulations v2019.5 (GROMACS) was employed. The simulation runs lasted for 100 ns for RLN-PLGF\u003csub\u003e1\u003c/sub\u003e and 200 ns for RLN-PLGF\u003csub\u003e3\u003c/sub\u003e and RLN-Fc. The CHARMM27 all-atom force field\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e was used for these simulations. Each system was placed in a cubic water box consisting of the TIP3P water model, which was neutralized with counter ions. The system was initially minimized using the steepest descent algorithm through 1000 steps to eliminate any unfavorable contacts. Subsequently, the equilibration processes were conducted using constant number, volume (NVT), and temperature (NPT). The protein backbone was constrained, while the solvent molecules and counter ions were allowed to move freely. The NVT was carried out for 100 ps at 300K. The NPT was executed for 100 ps at 1 bar. The restraints on heavy atom bonds were imposed using the LINCS algorithm.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e The Particle Mesh Ewald (PME)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e method was used to calculate long-range electrostatic interactions, with a cutoff value of 10 \u0026Aring; for short-range interactions. Periodic boundary conditions were implemented to avoid edge effects. The coordinate data were saved with a time step of 2 fs for every 1 ps. Finally, Visual Molecular Dynamics (VMD)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and Pymol\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e were used for the evaluation of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e8. Endosome isolation, characterization and lipidomics\u003c/h2\u003e \u003cp\u003eCells in culture were collected by centrifugation (850 g, 2 min, 4\u0026deg;C). Tissues were minced into small pieces, and subsequently homogenized using Dounce Homogenizer on ice. Cell lysis and organelles were then removed using lysosome enrichment kit according to the manufacturers\u0026rsquo; protocol. Endosome was further isolated by density gradient centrifugation for 145,000 g, 2 h at 4\u0026deg;C. The collected endosomes were washed three times and characterized using western blotting western with Rab-5, the biomarkers of endosome (Cell Signaling Technology). The isolated organelles were gone through lipidomics (\u003cb\u003eSupplementary Methods\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e9. Cells culture and assays\u003c/h2\u003e \u003cp\u003eCell lines were maintained in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Procell, China) and penicillin, streptomycin (Meilunbio, China). For activation of 3T3 cells, TGF-β1 was added at 10 ng/ml for 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003e9.1\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etransfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were plated in white, clear-bottom 96-well plates at 4000 cells per well for LX-2 cells and 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well for primary hepatocytes. On the second day, mLuc LNP was added to cells at 0.1\u0026micro;g mRNA per well. After 24 h incubation, the transfection efficiency was measured by Firefly-Glo Luciferase Reporter Assay Kit (Yeasen Biotechnology Co., Ltd.) following the manufacturer\u0026rsquo;s protocol, using BioTek synergy H1 microplate reader.\u003c/p\u003e \u003cp\u003e \u003cb\u003e9.2\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecell uptake\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrimary hepatocytes and LX-2 stellate cells were plated in 96-well plates at a density of ~\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells per well 24 h prior to the experiment. The cells were pre-incubated with small molecule endocytic inhibitors for 15 min. Small molecule endocytic inhibitors used in current study are listed below: cytochalasin D used for F-actin polymerization inhibition at 2.5\u0026micro;g/mL; methyl-β-cyclodextrin used for cholesterol/caveolae depletion at 2.5 mg/mL; nocodazole used for microtube inhibition at 5\u0026micro;g/mL; poly I used at the scavenger receptor inhibitor at 10 \u0026micro;g/mL; wortmannin used for inhibiting phosphoinositide pathway at 100 ng/mL and dynasore used for GTPase dynamin inhibition at 10 \u0026micro;g/mL. In addition, siRNA used for the knockdown of RBP-receptor, STRA6 was added at 200 nM 24 h at 37\u0026deg;C prior to LNP incubation. After pre-incubation with small molecules and siRNA, the supernatant was aspirated, washed twice with PBS. Then, 2.5 mol% BODIPY-lipid labeled LNPs (equivalent to 0.2 \u0026micro;g RNA per well) were added into each well of cells. After 2 h incubation at 37\u0026deg;C, the wells were washed 3 times with cold PBS and replaced with fresh media. The cellular uptake was determined by High Content Imaging and Analysis System (Cell Voyager CV8000, Yokogawa). Nucleus and lysosome were stained with Hoechst 33342 (1 \u0026micro;g/mL) and LysoTracker Red DND-99 (10000\u0026times; dilute) at 10 min before imaging. Flow cytometric analysis was performed 24 h after LNPs treatment and the mean fluorescence intensity was applied for quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e9.3 Live-cell imaging of mRNA release\u003c/h2\u003e \u003cp\u003eIntracellular release of mRNA from LNPs was visualized using an inverted Zeiss LSM880 confocal microscope. Airyscan array detector unit (Carl Zeiss AG) was used to strengthen and visualize the cytosol mRNA signals within the live cells. Five % BODIPY-lipid and 100% Cy-3-UTP replaced mRNA were applied for tracing the intracellular behavior of LNP and mRNA. Cells were treated with the labeled LNP with mRNA at a concentration of 2 \u0026micro;g/mL and incubated for 2 hours at 37\u0026deg;C. Subsequently, the media were replaced with fresh media containing 1 \u0026micro;g/mL Hoechst 33342, and endosome release was observed using a 100\u0026times; Plan-Neofluar 1.3 numerical aperture (NA) oil-immersion objective. For all experiments, the field of view (FOV) was set to 354, 25 \u0026micro;m \u0026times; 354, 25 \u0026micro;m (full). The pinhole was set to 1 Airy Unit (AU) for all channels. The acquired imaging data were processed using Zen2.3 software (blue edition) for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e10. Animal models\u003c/h2\u003e \u003cp\u003e All animal research was in compliance with ethical regulations approved by Peking University\u0026rsquo;s Institutional Animal Care and Use Committee. For models of CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis, 8-week-old male C57BL/6 mice were intraperitoneal injected with CCl\u003csub\u003e4\u003c/sub\u003e/olive oil (7/13, v/v, 50 \u0026micro;L per mouse) three times a week for a total of 4 or 6 weeks. For NASH models of C57BL/6 mice and golden hamster, 8-week-old male animals were fed with MCD diet for 8 weeks or CDHFD diet for 10 weeks. Fibrosis or NASH level were monitored by measuring serum AST and ALT levels.\u003c/p\u003e \u003cp\u003e \u003cb\u003e10.1\u003c/b\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eexpression\u003c/b\u003e\u003c/p\u003e \u003cp\u003emLuc LNPs were \u003cem\u003ei.v.\u003c/em\u003e administrated to healthy mice or mice with liver fibrosis (0.25 mg/kg). At 2, 6, 12 and 24 h after injection, bioluminescence imaging was performed using IVIS imaging system (Perkin Elmer). Hamsters with NASH were anesthetized by inhalation of isoflurane. A midline incision was made between the chin and sternum. The peripheral muscles were separated to expose the common carotid artery. LNPs at a dose of 0.25 mg/kg were injected through the carotid artery. Bioluminescence images were taken following the same process as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e10.2 mRNA delivery and expression in the tdTomato mice\u003c/h2\u003e \u003cp\u003eTo evaluate mRNA expression in different cell types, CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrotic LoxP-flanked tdTomato reporter mice were utilized. Total liver cells (including parenchymal and non-parenchymal cells) were harvested 48 h after \u003cem\u003ei.v.\u003c/em\u003e administration of mCre LNP formulations (1.5 mg/kg). Parenchymal cells, specifically primary hepatocytes were harvested by perfusion method as described above. Non-parenchymal cells (including endothelials, HSCs, leucoyctes) were harvested in the supernatant. Cell types were distinguished by size and specific markers by flow cytometry (Hepatocytes: low SSC/HSC, CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e; Endothelial: CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e, CD31\u003csup\u003e+\u003c/sup\u003e; Leucocytes, CD45\u003csup\u003e+\u003c/sup\u003e; other cells (mainly HSCs). The percentage of tdTomato\u003csup\u003e+\u003c/sup\u003e cells in each cell populations were measured and quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e10.3 Pharmacokinetics assay\u003c/h2\u003e \u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis models in C57BL/6 mice were developed as previously described. The mice were \u003cem\u003ei.v.\u003c/em\u003e treated with ATRA LNPs containing mRLN, mRLN-PLGF\u003csub\u003e1\u003c/sub\u003e, mRLN-PLGF\u003csub\u003e3\u003c/sub\u003e and mRLN-Fc at a concentration of 1.5 mg/kg. Subsequently, mice were euthanized at 0.25, 1, 3 and 6 days post injection, and both liver tissue and blood samples were harvested. The RLN content in the samples was determined using an ELISA kit (Boster bio) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e11. Statistics\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined using a two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test when only two value sets were compared or by ANOVA for comparison between multiple groups via GraphPad Prism 8.02. Exact \u003cem\u003eP\u003c/em\u003e values are documented in the figures or figure legends. Difference was considered to be significant if P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 unless otherwise indicated).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICTS OF INTERESTS\u003c/h2\u003e \u003cp\u003eL. M, X.Z.S., Z.Q.Z have filed a patent for the development of the described FORT strategy.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eL.M., X.Z.S., and Z.Q.Z. are responsible for all phases of the research. X.Z.S., Z.Q.Z., P.P.L., B.Y.L., P.X.Q., Y.Z.X., Z.H.Z., C.L.W. and B.M. performed experiments. Y.B.K. and H.Q.J. helped with SANS experiment. W.L. provided purified fusion proteins and cell lysates. W.Z.L. helped with cell uptake and confocal analysis. Q.W. participated in SPR and MST assay. J.Y. and Y.X.L. performed the molecular modeling and dynamics simulation of fusion proteins. Y.F.G. and L.S. performed the lipid membrane simulation. X.Z.S, Z.Q.Z. and L.M. wrote the manuscript. C.L., Y.L.Z., X.G.L., J.Q.L., X.D.X., D.D. and L.M. provided conceptual advice and supervised the study. All the authors discussed the results and assisted in the preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThis research was financially supported by National Key Research and Development Program of China (2023YFC3405000 to L.M), Beijing Natural Science Foundation (Z220022, to L.M), Beijing Municipal Science \u0026amp; Technology Commission (Z231100007223012 to L.M), the National Natural Science Foundation of China (NSFC) grants (HY2021-8, 82373807 to L. M., 82070460, 82270479, HY2021-1 to X.D.X). We thank the State Key Laboratory of Natural and Biomimetic Drugs, Peking University Biological Imaging Facility for confocal, animal, and tissue imaging services. The molecular modelling and dynamics simulations were carried out using the high-performance Linux cluster at the Computing and Data Science Core in the Chinese Institute for Brain Research, Beijing. We thank Keymed Biosciences for providing related proteins.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuby T, Gautier EL (2022) Immune cell-mediated features of non-alcoholic steatohepatitis. Nat Rev Immunol 22:429\u0026ndash;443\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMusso G, Cassader M, Gambino R (2016) Non-alcoholic steatohepatitis: emerging molecular targets and therapeutic strategies. 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Br J Pharmacol 174:921\u0026ndash;932\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"lipid nanoparticles, carboxyl retinoids, collagen binding domain, relaxin, NASH","lastPublishedDoi":"10.21203/rs.3.rs-3746897/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3746897/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibrotic diseases, like non-alcoholic steatohepatitis (NASH), pose challenges for targeted delivery and retention of therapeutic proteins due to increased extracellular matrix (ECM) deposition. Here we present a new approach to treat fibrotic diseases, termed \u0026ldquo;Fibrosis overexpression and retention (FORT)\u0026rdquo;. In this two-step strategy, we design 1) a retinoid derivative lipid nanoparticle (LNP) to enable specific mRNA overexpression in hepatic stellate cells, and 2) mRNA modifications which facilitate anchoring of therapeutic proteins in the fibrotic ECM. LNPs containing carboxyl retinoid derivatives, as opposed to alcohol or ester retinoid derivatives, effectively delivered mRNA, resulting in more than 10- fold enhancement of protein expression within the fibrotic liver. The carboxyl retinoid rearrangement on the LNP surface improved protein binding, sprouting, and membrane fusion. Therapeutic relaxin fusion proteins were then engineered with an endogenous collagen-binding domain. These fusion proteins exhibited increased retention in fibrotic lesions and reduced systemic side effects. \u003cem\u003eIn vivo\u003c/em\u003e, fibrosis-targeting LNPs encoding for mRNA fusion proteins demonstrated superior therapeutic efficacy in three clinically relevant NASH mouse models. This approach holds promise in chronic fibrotic diseases that are unsuited for direct injections of recombinant proteins.\u003c/p\u003e","manuscriptTitle":"RNA Nanotherapeutics with Fibrosis Overexpression and Retention (FORT) for NASH Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 06:48:58","doi":"10.21203/rs.3.rs-3746897/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26bdd59b-7da7-4447-b935-07b912feab50","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27869444,"name":"Biological sciences/Biotechnology/Biomaterials/Drug delivery"},{"id":27869445,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2024-08-28T07:05:30+00:00","versionOfRecord":{"articleIdentity":"rs-3746897","link":"https://doi.org/10.1038/s41467-024-51571-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-08-27 04:00:00","publishedOnDateReadable":"August 27th, 2024"},"versionCreatedAt":"2024-01-30 06:48:58","video":"","vorDoi":"10.1038/s41467-024-51571-8","vorDoiUrl":"https://doi.org/10.1038/s41467-024-51571-8","workflowStages":[]},"version":"v1","identity":"rs-3746897","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3746897","identity":"rs-3746897","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}