Deficiency of ASGR1 promotes liver injury by increasing GP73-mediated hepatic endoplasmic reticulum stress.

Liver injury is a core pathological process in the majority of liver diseases, yet the genetic factors predisposing individuals to its initiation and progression remain poorly understood. Here we show that asialoglycoprotein receptor 1 (ASGR1), a lectin specifically expressed in the liver, is downregulated in patients with liver fibrosis or cirrhosis and male mice with liver injury. ASGR1 deficiency exacerbates while its overexpression mitigates acetaminophen-induced acute and CCl4-induced chronic liver injuries in male mice. Mechanistically, ASGR1 binds to an endoplasmic reticulum stress mediator GP73 and facilitates its lysosomal degradation. ASGR1 depletion increases circulating GP73 levels and promotes the interaction between GP73 and BIP to activate endoplasmic reticulum stress, leading to liver injury. Neutralization of GP73 not only attenuates ASGR1 deficiency-induced liver injuries but also improves survival in mice received a lethal dose of acetaminophen. Collectively, these findings identify ASGR1 as a potential genetic determinant of susceptibility to liver injury and propose it as a therapeutic target for the treatment of liver injury.

Research progress on asialoglycoprotein receptor-targeted radiotracers designed for hepatic nuclear medicine imaging.

Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with ß-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian hepatocytes renders it an ideal hepatic-targeted biomarker. To date, ASGPR-targeted ligands have been actively developed for drug delivery and hepatic imaging. This review provides a comprehensive summary of the progress achieved to-date in the field of developing ASGPR-targeted nuclear medicine imaging (NMI) radiotracers, highlighting the recent advancements over the last decade in terms of structure, radionuclides and labeling strategies. The biodistribution patterns, imaging characteristics, challenges and future prospective are discussed.

Design, synthesis and biological evaluation of the positional isomers of the galactose conjugates able to target hepatocellular carcinoma cells via ASGPR-mediated cellular uptake and cytotoxicity.

Galactose as a recognizing motif for asialoglycoprotein receptor (ASGPR) is a widely accepted vector to deliver cytotoxic agents in the therapy of hepatocellular carcinoma (HCC), however, the individual hydroxyl group of galactose (Gal) contributed to recognizing ASGPR is obscure and remains largely unanswered in the design of glycoconjugates. Herein, we designed and synthesized five positional isomers of Gal-anthocyanin Cy5.0 conjugates and three Gal-doxorubicin (Dox) isomers, respectively. The fluorescence intensity of Gal-Cy5.0 conjugates accumulated in cancer cells hinted the optimal modification sites of positions C2 and C6. Comparing to the cytotoxicity of other conjugates, C2-Gal-Dox (11) was the most potent. Moreover, Gal-Dox conjugates significantly the toxicity of Dox. A progressively lower internalization capacity and siRNA technology implied the cellular uptake and cytotoxicity directly related to the ASGPR expression level. Accordingly, position C2 of galactose may be the best substitution site via ASGPR mediation in the design of anti-HCC glycoconjugates.

Serum IgA augments adhesiveness of cultured lung microvascular endothelial cells and suppresses angiogenesis.

Immunoglobulin A (IgA) is important in local immunity and is also abundant in the blood. This study aimed to evaluate the effects of serum IgA on cultured lung microvascular endothelial cells (HMVEC-Ls), which are involved in the pathogenesis of inflammatory lung diseases. Serum IgA induced adhesion molecules and inflammatory cytokine production from HMVEC-Ls, and enhanced adhesion of peripheral blood mononuclear cells to HMVEC-Ls. In contrast, migration, proliferation, and tube formation of HMVEC-Ls were significantly suppressed by serum IgA. Experiments with siRNAs and western blotting revealed that two known IgA receptors, ß1,4-galactosyltransferase 1 (b4GALT1) and asialoglycoprotein receptor 1 (ASGR1), and mitogen-activated protein kinase and nuclear factor-kappa B pathways were partly involved in serum IgA-induced cytokine production by HMVEC-Ls. Collectively, serum IgA enhanced cytokine production and adhesiveness of HMVEC-L, with b4GALT1 and ASGR1 partially being involved, and suppressed angiogenesis. Thus, serum IgA may be targeted to treat inflammatory lung diseases.

Synthesis, characterization, and biological verification of asialoglycoprotein receptor-targeted lipopolysaccharide-encapsulated PLGA nanoparticles for the establishment of a liver fibrosis animal model.

Liver fibrosis is generally preceded by various liver injuries and often leads to chronic liver diseases and even cirrhosis. Therefore, a liver fibrosis animal model is the cornerstone for the development of therapeutic strategies for hepatic diseases. Although administration of hepatotoxic substances and/or bile duct ligation have been widely performed to construct the in vivo model over the last decades, they are seriously hindered by time-consuming protocols, high mortality, and instability, indicating that an effective and safe approach for the induction of liver fibrosis is still urgently needed nowadays. In this study, we have developed asialoglycoprotein receptor (ASGPR)-targeted lipopolysaccharide (LPS)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles named ALPNPs for establishing an animal model of liver fibrosis. The ALPNPs are characterized as having a spherical nanostructure with size of 182.9 ± 8.89 nm and surface charge of -8.3 ± 1.48 mV. An anti-ASGPR antibody bound to the surface of the nanoparticles with a crosslinking efficiency of 95.03% allows ALPNPs to have hepatocyte-binding specificity. In comparison to free LPSs, the ALPNPs can induce higher aspartate aminotransferase and total bilirubin concentrations in plasma, reduce the blood flow rate in the portal system and the kidneys, and increase vascular resistance in the liver, kidneys, and collateral shunting vasculature. Based on histological and RNA-seq analyses, the ALPNPs can provide similar capability on inducing hepatic inflammation and fibrosis compared to free LPS but possess higher liver targetability than the naked drug. In addition, the ALPNPs are less toxic in organs other than the liver in comparison to free LPS, demonstrating that the ALPNPs do not elicit off-target effects in vivo. Given the aforementioned efficacies with other merits such as biocompatibility and drug release controllability provided by PLGA, we anticipate that the developed ALPNPs are highly applicable in establishing animal models of liver fibrosis in pre-clinical studies.

Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Targeted delivery of oligonucleotides using multivalent protein-carbohydrate interactions.

Cell surface protein-carbohydrate interactions are essential for tissue-specific recognition and endocytosis of viruses, some bacteria and their toxins, and many glycoproteins. Often protein-carbohydrate interactions are multivalent - multiple copies of glycans bind simultaneously to multimeric receptors. Multivalency enhances both affinity and binding specificity, and is of interest for targeted delivery of drugs to specific cell types. The first such example of carbohydrate-mediated drug delivery to reach the clinic is Givosiran, a small interfering ribonucleic acid (siRNA) that is conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand. This ligand enables efficient uptake of the nucleic acid by the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Synthetic multivalent ligands for ASGP-R were among the first 'cluster glycosides' developed at the birth of multivalent glycoscience around 40 years ago. In this review we trace the history of 'GalNAc targeting' from early academic studies to current pharmaceuticals and consider what other opportunities could follow the success of this delivery technology.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Liver cancer cells as the model for developing liver-targeted RNAi therapeutics.

RNAi is a sequence-specific gene regulation mechanism that involves small interfering RNAs (siRNAs). RNAi therapeutic has become a new class of precision medicine and has shown great potential in treating liver-associated diseases, especially metabolic diseases. To facilitate the development of liver-targeted RNAi therapeutics in cell model, we surveyed a panel of liver cancer cell lines for the expression of genes implicated in RNAi therapeutics including the asialoglycoprotein receptor (ASGR) and metabolic disease associated genes PCSK9, ANGPTL3, CIDEB, and LDLR. A high-content screen assay based on lipid droplet staining confirmed the involvement of PCSK9, ANGPTL3, and CIDEB in lipid metabolism in selected liver cancer cell lines. Several liver cancer cell lines have high levels of ASGR1 expression, which is required for liver-specific uptake of GalNAc-conjugated siRNA, a clinically approved siRNA delivery platform. Using an EGFP reporter system, we demonstrated Hep G2 can be used to evaluate gene knockdown efficiency of GalNAc-siRNA. Our findings pave the way for using liver cancer cells as a convenient model system for the identification and testing of siRNA drug candidate genes and for studying ASGR-mediated GalNAc-siRNA delivery in liver.

Comprehensive analysis of SARS-CoV-2 receptor proteins in human respiratory tissues identifies alveolar macrophages as potential virus entry site.

AIMS: COVID-19 has had enormous consequences on global health-care and has resulted in millions of fatalities. The exact mechanism and site of SARS-CoV-2 entry into the body remains insufficiently understood. Recently, novel virus receptors were identified, and alveolar macrophages were suggested as a potential viral entry cell type and vector for intra-alveolar virus transmission. Here, we investigated the protein expression of 10 well-known and novel virus entry molecules along potential entry sites in humans using immunohistochemistry. METHODS AND RESULTS: Samples of different anatomical sites from up to 93 patients were incorporated into tissue microarrays. Protein expression of ACE2, TMPRSS2, furin, CD147, C-type lectin receptors (CD169, CD209, CD299), neuropilin-1, ASGR1 and KREMEN1 were analysed. In lung tissues, at least one of the three receptors ACE2, ASGR1 or KREMEN1 was expressed in the majority of cases. Moreover, all the investigated molecules were found to be expressed in alveolar macrophages, and co-localisation with SARS-CoV-2 N-protein was demonstrated using dual immunohistochemistry in lung tissue from a COVID-19 autopsy. While CD169 and CD209 showed consistent protein expression in sinonasal, conjunctival and bronchiolar tissues, neuropilin-1 and ASGR1 were mostly absent, suggesting a minor relevance of these two molecules at these specific sites. CONCLUSION: Our results extend recent discoveries indicating a role for these molecules in virus entry at different anatomical sites. Moreover, they support the notion of alveolar macrophages being a potential entry cell for SARS-CoV-2.

Sorafenib-Entrapped, Self-Assembled Pullulan-Stearic Acid Biopolymer-Derived Drug Delivery System to PLC/PRF/5 Hepatocellular Carcinoma Model.

Purpose: This study aimed to design a prototypic drug delivery system (DDS) made of an amphiphilic, pullulan (Pull)-derived biodegradable polymer for targeting the asialoglycoprotein receptor (ASGPR) overexpressed in HCC. Stearic acid (SA) was conjugated to increase the hydrophobicity of pullulan (Pull-SA). Methods: Pullulan (Pull) was linked to stearic acid (SA) after functional group modifications via EDC/NHS chemistry and characterized. Sorafenib tosylate (SRFT) was entrapped in pullulan-stearic acid nanoparticles (Pull-SA-SRFT) and its particle size, zeta potential, entrapment efficiency (EE), loading capacity (LC), and release efficiency was measured. The competence of Pull-SA-SRFT over SRFT in vitro was assessed using the ASGPR over-expressing PLC/PRF/5 hepatocellular carcinoma (HCC) cell line. This was done by studying cytotoxicity by MTT assay and chromosome condensation assay, early apoptosis by annexin-Pi staining, and late apoptosis by live-dead assay. The cellular uptake study was performed by incorporating coumarin-6 (C6) fluorophore in place of SRFT in Pull-SA conjugates. A biodistribution study was conducted in Swiss-albino mice to assess the biocompatibility and targeting properties of SRFT and Pull-SA-SRFT to the liver and other organs at 1, 6, 24, and 48 h. Results: The characterization studies of the copolymer confirmed the successful conjugation of Pull-SA. The self-assembled amphiphilic nanocarrier could proficiently entrap the hydrophobic drug SRFT to obtain an entrapment efficiency of 95.6% (Pull-SA-SRFT). Characterization of the synthesized nanoparticles exhibited highly desirable nanoparticle characteristics. In vitro, apoptotic studies urged that Pull-SA-SRFT nanoparticle was delivered more efficiently to HCC than SRFT. The cellular uptake study performed, gave propitious results in 4 hrs. The biodistribution study conducted in immunocompetent mice suggested that Pull-SA-SRFT was delivered more than SRFT to the liver when compared to other organs, and that the system was biocompatible. Conclusion: Pull-SA-SRFT is a promisingly safe, biodegradable, cell-specific nanocarrier and a potential candidate to target hydrophobic drugs to HCC.

Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with CNS inflammation in children.

Fetuin-A is a liver derived plasma protein showing highest serum concentrations in utero, preterm infants, and neonates. Fetuin-A is also present in cerebrospinal fluid (CSF). The origin of CSF fetuin-A, blood-derived via the blood-CSF barrier or synthesized intrathecally, is presently unclear. Fetuin-A prevents ectopic calcification by stabilizing calcium and phosphate as colloidal calciprotein particles mediating their transport and clearance. Thus, fetuin-A plays a suppressive role in inflammation. Fetuin-A is a negative acute-phase protein under investigation as a biomarker for multiple sclerosis (MS). Here we studied the association of pediatric inflammatory CNS diseases with fetuin-A glycosylation and phosphorylation. Paired blood and CSF samples from 66 children were included in the study. Concentration measurements were performed using a commercial human fetuin-A/AHSG ELISA. Of 60 pairs, 23 pairs were analyzed by SDS-PAGE following glycosidase digestion with PNGase-F and Sialidase-AU. Phosphorylation was analyzed in 43 pairs by Phos-TagTM acrylamide electrophoresis following alkaline phosphatase digestion. Mean serum and CSF fetuin-A levels were 0.30 ± 0.06 mg/ml and 0.644 ± 0.55 µg/ml, respectively. This study showed that serum fetuin-A levels decreased in inflammation corroborating its role as a negative acute-phase protein. Blood-CSF barrier disruption was associated with elevated fetuin-A in CSF. A strong positive correlation was found between the CSF fetuin-A/serum fetuin-A quotient and the CSF albumin/serum albumin quotient, suggesting predominantly transport across the blood-CSF barrier rather than intrathecal fetuin-A synthesis. Sialidase digestion showed increased asialofetuin-A levels in serum and CSF samples from children with neuroinflammatory diseases. Desialylation enhanced hepatic fetuin-A clearance via the asialoglycoprotein receptor thus rapidly reducing serum levels during inflammation. Phosphorylation of fetuin-A was more abundant in serum samples than in CSF, suggesting that phosphorylation may regulate fetuin-A influx into the CNS. These results may help establish Fetuin-A as a potential biomarker for neuroinflammatory diseases.

A glutathione activatable bioprobe for detection of hepatocellular carcinoma cells in peripheral blood via carbohydrate-protein interaction.

Due to high recurrence and metastasis rates leading to high mortality of hepatocellular carcinoma (HCC), detection of HCC circulating tumor cells (HCC-CTCs), which are regarded as an HCC blood marker, holds great significance in HCC early diagnosis, metastasis evolution, and prognosis. However, current existing circulating tumor cell (CTC) detection methods require multiple steps, and have low accuracy due to extremely rare CTCs in peripheral blood (PB). Thus, a simple and sensitive HCC-CTCs detection method is urgently needed. Here, a glutathione (GSH) activatable bioprobe (LacCC) targeting HCC cells was first developed through coordinating copper ions (Cu2+) to lactose modified coumarin derivative (LacC). Owing to the carbohydrate-protein interaction between lactose group and asialoglycoprotein receptors (ASGPRs) overexpressed on the membrane of HCC cells, LacCC displays selectivity towards HCC cells. The fluorescence of LacCC recovers rapidly within 2 min upon demetallation by high concentration of GSH in HCC cells. In simulated PB samples, as low as 10 HepG2 cells were detected via CLSM after removing red blood cells (RBCs) and culturing with LacCC. By coupling with flow cytometry, LacCC can achieve quantitative detection of HCC cells with low detection limit (LOD) of 3 cells per sample. Thus, this bioprobe possessing ASGPRs targetability and fast GSH responsiveness shows ultrasensitive detectability towards HCC cells in PB, which may have the potential for simple yet highly sensitive HCC-CTCs detection.

Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion.

High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3.

Liver cancer is characterized by aggressive growth and high mortality. Asialoglycoprotein receptor 1 (ASGR1), which is expressed almost exclusively in liver cells, is reduced in liver cancer. However, the specific mechanism of ASGR1 function in liver cancer has not been fully elucidated. On the basis of database screening, we identified ASGR1 as a tumor suppressor regulated by DNA methylation. Expression of ASGR1 was downregulated in liver cancer and correlated with tumor size, grade, and survival. Functional gain and loss experiments showed that ASGR1 suppresses the progression of liver cancer in vivo and in vitro. RNA sequencing and mass spectrometry showed that ASGR1 inhibits tyrosine phosphorylation of STAT3 by interacting with Nemo-like kinase (NLK). NLK bound the SH2 domain of STAT3 in an ATP-dependent manner and competed with glycoprotein 130 (GP130), ultimately suppressing GP130/JAK1-mediated phosphorylation of STAT3. ASGR1 altered the binding strength of NLK and STAT3 by interacting with GP130. Furthermore, the domain region of NLK was crucial for binding STAT3 and curbing its phosphorylation. Collectively, these results confirm that ASGR1 suppresses the progression of liver cancer by promoting the binding of NLK to STAT3 and inhibiting STAT3 phosphorylation, suggesting that approaches to activate the ASGR1-NLK axis may be a potential therapeutic strategy in this disease. SIGNIFICANCE: ASGR1 downregulation by DNA methylation facilitates liver tumorigenesis by increasing STAT3 phosphorylation.

Effect of hydrophile-lipophile balance of the linker in Gal/GalNAc ligands on high-affinity binding of galactosylated liposomes by the asialoglycoprotein receptor.

In this study, we explored the effect of the hydrophile-lipophile balance (HLB) in the linker unit of Galactose (Gal)/N-acetylgalactosamine (GalNAc) ligands on their affinity toward asialoglycoprotein receptors (ASGPRs). Two Gal/GalNAc ligands with lipophilic linkers-{(5-cholesten-3b-ol)[(2-acetamido-2-deoxy-d-galactopyranose-6-o)sebacate]} (CHS-6-GalNAc) and {(5-cholesten-3b-ol)[(d-galactopyranose-6-o)sebacate]} (CHS-6-Gal)-and two with hydrophilic linkers-{(5-cholesten-yl)[(4-O-b-D-galactopyranosyl)-D-glucitol-6-yl]sebacate} (CHS-1-Gal) and {(5-cholesten-3a-ol)[(2-acetamido-2-deoxy-d-galactopyranose-6-o)3,6-dioxa-octanedioate]} (CHS-PEG2-6-GalNAc)-were synthesized by enzymatic catalysis. Compared with unmodified liposomes, all Gal/GalNAc ligand-modified liposomes showed higher efficiency toward the hepatocyte target as evaluated by weighted-average overall drug-targeting efficiency (Te*) in vivo and HepG2 cell uptake efficiency in vitro. The ligands containing linkers with high HLB values (i.e., CHS-PEG2-6-GalNAc and CHS-1-Gal) exhibited higher ASGPR affinity than those containing linkers with low HLB values (i.e., CHS-6-GalNAc and CHS-6-Gal). We used molecular-dynamics (MD) simulations to investigate the structure-activity relationship between the HLB value of the linker in a ligand and ASGPR affinity. MD simulation results indicated that a Gal/GalNAc ligand with a more hydrophilic linker (i.e., higher HLB value) unit tended to have a higher solvent-accessible surface area (SASA), leading to lower steric hindrance for effective ASGPR recognition. The results of this study will provide an improved design for Gal/GalNAc ligand-based surface-modified liposomes with high ASGPR affinity.

SARS-CoV-2 cell entry beyond the ACE2 receptor.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is known as the major viral entry site for SARS-CoV-2. However, viral tissue tropism and high rate of infectivity do not directly correspond with the level of ACE2 expression in the organs. It may suggest involvement of other receptors or accessory membrane proteins in SARSCoV-2 cell entry. METHODS AND RESULTS: A systematic search was carried out in PubMed/Medline, EMBASE, and Cochrane Library for studies reporting SARS-CoV-2 cell entry. We used a group of the MeSH terms including "cell entry", "surface receptor", "ACE2", and "SARS-CoV-2". We reviewed all selected papers published in English up to end of February 2022. We found several receptors or auxiliary membrane proteins (including CD147, NRP-1, CD26, AGTR2, Band3, KREMEN1, ASGR1, ANP, TMEM30A, CLEC4G, and LDLRAD3) along with ACE2 that facilitate virus entry and transmission. Expression of Band3 protein on the surface of erythrocytes and evidence of binding with S protein of SARS-CoV-2 may explain asymptomatic hypoxemia during COVID19 infection. The variants of SARS-CoV-2 including the B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.2+ (Delta+), and B.1.1.529 (Omicron) may have different potency to bond with these receptors. CONCLUSIONS: The high rate of infectivity of SARS-CoV-2 may be due to its ability to enter the host cell through a group of cell surface receptors. These receptors are potential targets to develop novel therapeutic agents for SARS-CoV-2.

Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes.

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3'-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3'-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization.

Generation of a human iPSC line CIBi010-A with a reporter for ASGR1 using CRISPR/Cas9.

ASGR1 is a liver-specific surface marker that has been used to purify human pluripotent stem cell (PSC)-derived hepatocytes (iHeps). Furthermore, ASGR1+ iHeps represents a more mature subpopulation of iHeps. To utilize this marker for optimizing iHep differentiation and purification, we substituted the stop coden of ASGR1 with a fluorescent reporter protein mCherry in a human iPSC line iPSN0052 via CRISPR/Cas9-mediated homologus recombination. The generated CIBi010-A enableds us to monitor ASGR1 expression during hepatic differentiation and thus can be used to optimize our hepatic differentiation procedures.

New and effective method to develop primary hepatocytes from liver cancer patients.

Liver cancer (LC) is one of the most common malignant tumors worldwide. Since the mechanism of LC pathogenesis and metastasis cannot be carried out directly on the human body, it is particularly important to establish human liver cancer cell lines for research in vitro. In this study, tissue block adherence method combined with cell clumps digestion method was used to establish primary human hepatocytes (PHHs) with a successful rate of 60% (45/75). Short tandem repeat (STR) analysis proved the cells were derived from its paired tissues. These cells from hepatocellular carcinoma (HCC) expressed NTCP and secreted ALB and AAT as detected by western blot, and expressed hepatocyte-specific membrane protein ASGR1 as detected by flow cytometry. Liver cancer biomarkers like CK7 in ICC (intrahepatic cholangiocarcinoma), AFP, and GPC3 in HCC expressed of different degree as detected by immunohistochemical analysis. These cells displayed typical liver cancer cell morphological characteristics and can passage stably. In conclusion, we developed an effective method to establish PHHs. Further studies are necessary to study if these cells maintaining other liver function and reproduce the physiology of the tumors and how these cells behavior in the drug development.