Search

The formulation of novel delivery protocols for the targeted delivery of genes into hepatocytes by receptor mediation is important for the treatment of liver-specific disorders, including cancer. Non-viral delivery methods have been extensively studied for gene therapy. Gold nanoparticles (AuNPs) have gained attention in nanomedicine due to their biocompatibility. In this study, AuNPs were synthesized and coated with polymers: chitosan (CS), and polyethylene glycol (PEG). The targeting moiety, lactobionic acid (LA), was added for hepatocyte-specific delivery. Physicochemical characterization revealed that all nano-formulations were spherical and monodispersed, with hydrodynamic sizes between 70 and 250 nm. Nanocomplexes with pCMV-Luc DNA (pDNA) confirmed that the NPs could bind, compact, and protect the pDNA from nuclease degradation. Cytotoxicity studies revealed that the AuNPs were well tolerated (cell viabilities > 70%) in human hepatocellular carcinoma (HepG2), embryonic kidney (HEK293), and colorectal adenocarcinoma (Caco-2) cells, with enhanced transgene activity in all cells. The inclusion of LA in the NP formulation was notable in the HepG2 cells, which overexpress the asialoglycoprotein receptor on their cell surface. A five-fold increase in luciferase gene expression was evident for the LA-targeted AuNPs compared to the non-targeted AuNPs. These AuNPs have shown potential as safe and suitable targeted delivery vehicles for liver-directed gene therapy.

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., (© 2024. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells., (© 2024. The Author(s).)

Background: Asialoglycoprotein receptor 1 (ASGR1), primarily expressed on hepatocytes, promotes the clearance and the degradation of glycoproteins, including lipoproteins, from the circulation. In humans, loss-of-function variants of ASGR1 are associated with a favorable metabolic profile and reduced incidence of cardiovascular diseases. The molecular mechanisms by which ASGR1 could affect the onset of metabolic syndrome and obesity are unclear. Therefore, here we investigated the contribution of ASGR1 in the development of metabolic syndrome and obesity., Methods: ASGR1 deficient mice (ASGR1 -/- ) were subjected to a high-fat diet (45% Kcal from fat) for 20 weeks. The systemic metabolic profile, hepatic and visceral adipose tissue were characterized for metabolic and structural alterations, as well as for immune cells infiltration., Results: ASGR1 -/- mice present a hypertrophic adipose tissue with 41% increase in fat accumulation in visceral adipose tissue (VAT), alongside with alteration in lipid metabolic pathways. Intriguingly, ASGR1 -/- mice exhibit a comparable response to an acute glucose and insulin challenge in circulation, coupled with notably decreased in circulating cholesterol levels. Although the liver of ASGR1 -/- have similar lipid accumulation to the WT mice, they present elevated levels of liver inflammation and a decrease in mitochondrial function., Conclusion: ASGR1 deficiency impacts energetic homeostasis during obesity leading to improved plasma lipid levels but increased VAT lipid accumulation and liver damage., (© 2024. The Author(s).)

Cationic glycopolymers stand out as gene delivery nanosystems due to their inherent biocompatibility and high binding affinity to the asialoglycoprotein receptor (ASGPR), a target receptor overexpressed in hepatocellular carcinoma (HCC) cells. However, their synthesis procedure remains laborious and complex, with problems of solubilization and the need for protection/deprotection steps. Here, a mini-library of well-defined poly(2-aminoethyl methacrylate hydrochloride- co -poly(2-lactobionamidoethyl methacrylate) (PAMA- co -PLAMA) glycopolymers was synthesized by activators regenerated by electron transfer (ARGET) ATRP to develop an efficient gene delivery nanosystem. The glycoplexes generated had suitable physicochemical properties and showed high ASGPR specificity and high transfection efficiency. Moreover, the HSV-TK/GCV suicide gene therapy strategy, mediated by PAMA 144 - co -PLAMA 19 -based nanocarriers, resulted in high antitumor activity in 2D and 3D culture models of HCC, which was significantly enhanced by the combination with small amounts of docetaxel. Overall, our results demonstrated the potential of primary-amine polymethacrylate-containing-glycopolymers as HCC-targeted suicide gene delivery nanosystems and highlight the importance of combined strategies for HCC treatment.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (©2022 American Association for Cancer Research.)

High cholesterol is a major risk factor for cardiovascular disease 1 . 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 disease 2 . ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins 3 . 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 faeces 4 , 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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Background: The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its N -linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in hemophilia A (HA) is unknown., Objective: To investigate the variability in FVIII PK outcomes in relation to genetic variation in the ASGR2, encoding the ASGPR2 subunit., Methods: Thirty-two HA patients with FVIII:C ≤2 IU/dL underwent 66 single-dose FVIII PK studies. PK parameters were evaluated in relation to ASGR2 5' untranslated region (5'UTR) polymorphisms, which were investigated by recombinant and white blood cell reverse transcription-polymerase chain reaction approaches., Results: The 5'UTR polymorphisms determine a frequent and conserved haplotype (HT1) in a regulatory region. The HT1 homozygotes may differ in the amounts of alternatively spliced mRNA transcripts and thus ASGPR2 isoforms. Compared with the other ASGR2 genotypes, the c.-95TT homozygotes ( n  = 9), showed threefold longer Alpha HL (3.60 hours, 95% confidence interval: 1.44-5.76, p  = 0.006), and the c.-95TC heterozygotes ( n  = 17) showed 25% shorter mean residence time (MRT; 18.5 hours, 15.0-22.0, p  = 0.038) and 32% shorter Beta HL (13.5 hours, 10.9-16.0, p  = 0.016). These differences were confirmed in patients ( n  = 27) undergoing PK studies ( n  = 54) with full-length FVIII only. In different linear regression models, the contribution of the ASGR2 genotypes remained significant after adjustment by ABO genotypes and von Willebrand factor (VWF) antigen levels, and explained 14% (MRT), 15 to 18% (Beta HL), and 22% (Alpha HL) of parameter variability., Conclusion: Infused FVIII distribution was modulated by frequent ASGR2 genotypes, independently from and together with ABO and VWF antigen levels, which has potential implications for genetically tailored substitutive treatment in HA., Competing Interests: M. Morfini reports personal fees from Kedrion, grants from Pfizer, personal fees from Novo Nordisk, personal fees from Sobi, personal fees from Bayer, personal fees from Bioverativ, personal fees from Octapharma, personal fees from CSL Behring, outside the submitted work. D. Balestra reports grants and personal fees from Bayer. A. Branchini reports grants and personal fees from Pfizer, grants from Bayer, grants and nonfinancial support from Grifols, outside the submitted work. G. Castaman reports personal fees from Roche, personal fees from Bayer, personal fees from Shire, grants and personal fees from CSL Behring, grants and personal fees from Sobi, personal fees from Uniqure, grants from Pfizer, personal fees from Kedrion, personal fees from Novo Nordisk, personal fees from Werfen, outside the submitted work. F. Bernardi reports grants from Bayer, during the conduct of the study; grants from Pfizer, outside the submitted work. The other authors state that they have no conflict of interest., (Thieme. All rights reserved.)

Ligand-targeted drug delivery (LTDD) has gained more attention in the field of nucleic acid therapeutics. To further elicit the potential of therapeutic oligonucleotides by means of LTDD, we newly developed ( R )- and ( S )-3-amino-1,2-propanediol (APD) manifold for ligand conjugation. N -acetylgalactosamine (GalNAc)/asialoglycoprotein receptor (ASGPr) system has been shown to be a powerful and robust paradigm of LTDD. Our novel APD-based GalNAc (GalNAc APD ) was shown to have intrinsic chemical instability that could play a role in better manipulation of active drug release. The APD manifold also enables facile production of conjugates through an on-support ligand cluster synthesis. We showed in a series of in vivo studies that while the knockdown activity of antisense oligonucleotides (ASOs) bearing 5'-GalNAc APD was comparable to the conventional hydroxy-L-prolinol-linked GalNAc (GalNAc HP ), 3'-GalNAc APD elicited ASO activity by more than twice as much as the conventional 3'-GalNAc HP . This was ascribed partly to the GalNAc APD 's ideal susceptibility to nucleolytic digestion, which is expected to facilitate cytosolic internalization of ASO drugs. Moreover, an in vivo / ex vivo imaging study visualized the enhancement effect of monoantennary GalNAc APD on liver localization of ASOs. This versatile manifold with chemical and biological instability would benefit therapeutic oligonucleotides that target both the liver and extrahepatic tissues.

Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact., Competing Interests: The authors have declared that no competing interests exist.

A population genetic study identified that the asialoglycoprotein receptor 1 (ASGR1) mutation carriers had substantially lower non-HDL-cholesterol (non-HDL-c) levels and reduced risks of cardiovascular diseases. However, the mechanism behind this phenomenon remained unclear. Here, we established Asgr1-knockout mice that represented a plasma lipid profile with significantly lower non-HDL-c and triglyceride (TG) caused by decreased secretion and increased uptake of VLDL/LDL. These 2 phenotypes were linked with the decreased expression of microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9, 2 key targeted genes of sterol regulatory element-binding proteins (SREBPs). Furthermore, there were fewer nuclear SREBPs (nSREBPs) on account of more SREBPs being trapped in endoplasmic reticulum, which was caused by an increased expression of insulin-induced gene 1 (INSIG1), an anchor of SREBPs. Overexpression and gene knockdown interventions, in different models, were conducted to rescue the ASGR1-deficient phenotypes, and we found that INSIG1 knockdown independently reversed the ASGR1-mutated phenotypes with increased serum total cholesterol, LDL-c, TG, and liver cholesterol content accompanied by restored SREBP signaling. ASGR1 rescue experiments reduced INSIG1 and restored the SREBP network defect as manifested by improved apolipoprotein B secretion and reduced LDL uptake. Our observation demonstrated that increased INSIG1 is a critical factor responsible for ASGR1 deficiency-associated lipid profile changes and nSREBP suppression. This finding of an ASGR1/INSIG1/SREBP axis regulating lipid hemostasis may provide multiple potential targets for lipid-lowering drug development.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9 -/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK 103 ↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. R. C. A. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Amgen Canada Research Chair in the Division of Nephrology at St. Joseph’s Healthcare and McMaster University., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Serum protein interactions are evaluated during the drug development process since they determine the free drug concentration in blood and thereby can influence the drug's pharmacokinetic and pharmacodynamic properties. While the impact of serum proteins on the disposition of small molecules is well understood, it is not yet well characterized for a new modality, RNA interference therapeutics. When administered systemically, small interfering RNAs (siRNAs) conjugated to the N -acetylgalactosamine (GalNAc) ligand bind to proteins present in circulation. However, it is not known if these protein interactions may impact the GalNAc-conjugated siRNA uptake into hepatocytes mediated through the asialoglycoprotein receptor ( ASGPR ) and thereby influence the activity of GalNAc-conjugated siRNAs. In this study, we assess the impact of serum proteins on the uptake and activity of GalNAc-conjugated siRNAs in primary human hepatocytes. We found that a significant portion of the GalNAc-conjugated siRNAs is bound to serum proteins. However, ASGPR -mediated uptake and activity of GalNAc-conjugated siRNAs were minimally impacted by the presence of serum relative to their uptake and activity in the absence of serum. Therefore, in contrast to small molecules, serum proteins are expected to have minimal impact on pharmacokinetic and pharmacodynamic properties of GalNAc-conjugated siRNAs.

Background and Aims: Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes., Approach and Results: Recent work provided evidence for the control of TPO mRNA expression in liver and bone marrow (BM) by scanning circulating platelets. The Ashwell-Morell receptor (AMR) was identified to bind desialylated platelets to regulate hepatic thrombopoietin (TPO) production by Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) activation. Two-thirds partial hepatectomy (PHx) was performed in mice. Platelet activation and clearance by AMR/JAK2/STAT3 signaling and TPO production were analyzed at different time points after PHx. Here, we demonstrate that PHx in mice led to thrombocytopenia and platelet activation defects leading to bleeding complications, but unaltered arterial thrombosis, in these mice. Platelet counts were rapidly restored by up-regulation and crosstalk of the AMR and the IL-6 receptor (IL-6R) to induce JAK2-STAT3-TPO activation in the liver, accompanied by an increased number of megakaryocytes in spleen and BM before liver was completely regenerated., Conclusions: The AMR/IL-6R-STAT3-TPO signaling pathway is an acute-phase response to liver injury to reconstitute hemostasis. Bleeding complications were attributable to thrombocytopenia and platelet defects induced by elevated PGI 2 , NO, and bile acid plasma levels early after PHx that might also be causative for the high mortality in patients with liver disease., (© 2020 by the American Association for the Study of Liver Diseases.)

Background: The decrease in asialoglycoprotein receptor (ASGPR) levels is observed in patients with chronic liver disease and liver tumor. The aim of our study was to develop ASGPR-targeted superparamagnetic perfluorooctylbromide nanoparticles (M-PFONP) and wonder whether this composite agent could target buffalo rat liver (BRL) cells in vitro and could improve R 2 ∗ value of the rat liver parenchyma after its injection in vivo., Methods: GalPLL, a ligand of ASGPR, was synthesized by reductive amination. ASGPR-targeted M-PFOBNP was prepared by a film hydration method coupled with sonication. Several analytical methods were used to investigate the characterization and safety of the contrast agent in vitro. The in vivo MR T 2 ∗ mapping was performed to evaluate the enhancement effect in rat liver., Results: The optimum concentration of Fe 3 O 4 nanoparticles inclusion in GalPLL/M-PFOBNP was about 52.79  µ g/mL, and the mean size was 285.6 ± 4.6 nm. The specificity of GalPLL/M-PFOBNP for ASGPR was confirmed by incubation experiment with fluorescence microscopy. The methyl thiazolyl tetrazolium (MTT) test showed that there was no significant difference in the optical density (OD) of cells incubated with all GalPLL/M-PFOBNP concentrations. Compared with M-PFOBNP, the increase in R 2 ∗ value of the rat liver parenchyma after GalPLL/M-PFOBNP injection was higher., Conclusions: GalPLL/M-PFOBNP may potentially serve as a liver-targeted contrast agent for MR receptor imaging., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Yang Li et al.)

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig ( Sus scrofa domestica ) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1 , CMAH , β4GalNT2 , vWF , ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.

Background: Gastric cancer (GC) is the fourth most common cause of cancer-related deaths in the world and 5-year overall survival (OS) rate is less than 10%. So, it is urgent to identified novel diagnostic and prognostic biomarkers., Methods: Twelve GEO (gene expression omnibus) datasets were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between GC and normal tissues were screened and integrated using limma and RobustRankAggreg (RRA) packages in R software. Protein-protein interaction (PPI) network, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses for DEGs were conducted via STRING and DAVID, respectively. Moreover, Cox regression model was used to construct a gene prognosis signature., Results: Ten genes (COL1A1, CXCL8, COL3A1, SPP1, COL1A2, TIMP1, CXCL1, BGN, MMP3 and SERPINE1) were identified and might be highly related to GC. Further analysis showed high expression of CXCL8, COL3A1, CXCL1, MMP3 and SERPINE1, were significantly associated with late stage of GC. Lastly, we build a seven-gene prognosis signature (CYP19A1, SERPINE1, CGB5, CALCR, ASGR2, CYTL1 and ABCB5), which can give a good prediction of OS., Conclusions: Our article screened out key genes highly associating with GC's developments and prognosis, and it is useful for researcher to further understand GC's molecular basis and direct the synthesis medicine of GC.

House dust mite (HDM) is a major allergen that causes allergic diseases such as atopic dermatitis. However, the regulatory mechanisms of HDM-induced immune responses are incompletely understood. NC/Nga mice are an inbred strain that is more susceptible to HDM and develops more severe dermatitis than other strains. Using whole-exome sequencing, we found that NC/Nga mice carry a stop-gain mutation in Clec10a , which encodes a C-type lectin receptor, Clec10a (MGL1/CD301a). The repair of this gene mutation using the CRISPR-Cas9 system ameliorated HDM-induced dermatitis, indicating that the Clec10a mutation is responsible for hypersensitivity to HDM in NC/Nga mice. Similarly, Clec10a -/- mice on the C57BL/6J background showed exacerbated HDM-induced dermatitis. Clec10a expressed on skin macrophages inhibits HDM-induced Toll-like receptor 4 (TLR4)-mediated inflammatory cytokine production through the inhibitory immunoreceptor tyrosine activating motif in its cytoplasmic portion. We identified asialoglycoprotein receptor 1 (Asgr1) as a functional homolog of mouse Clec10a in humans. Moreover, we found that a mucin-like molecule in HDM is a ligand for mouse Clec10a and human Asgr1. Skin application of the ligand ameliorated a TLR4 ligand-induced dermatitis in mice. Our findings suggest that Clec10a in mice and Asgr1 in humans play an important role in skin homeostasis against inflammation associated with HDM-induced dermatitis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The ASGR1 was recently shown to play a key role in the development of coronary artery disease (CAD), but its exact mechanism of action in the CAD pathogenesis is not yet known. This study evaluates the possible association between the expression level of ASGR1 and its downstream transcription factor FOXM1 in the inflammatory cells of peripheral blood (PBMC) and the pathogenesis of CAD in the Diabetic condition., Methods: Blood samples were taken from the candidates who had visited the Tehran Heart Center and had underwent diagnostic tests with respect to diabetes and CAD. The peripheral blood cells were harvested, RNA was extracted, and cDNA was synthesized. The qRT-PCR was performed on 79 cDNA samples taken from 49 CAD + patients and 30 CAD - patients., Results: In this study, we observed a significant decrease of ASGR1 expression in the PBMC of CAD + patients compared to the CAD - patients. We did not identify any considerable differences in the expression of FOXM1 in patients' subgroups with respect to the diabetes and CAD., Conclusion: The results of our study determine the association of ASGR1 expression and CAD pathogenesis. However, we do not know whether this result is the cause or the effect of CAD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Advances in medicinal chemistry have produced new chemical classes of antisense oligonucleotides (ASOs) with enhanced therapeutic properties. Conjugation of the triantennary N-acetylgalactosamine (GalNAc 3 ) moiety to the extensively characterized phosphorothioate (PS)-modified 2'-O-methoxyethyl (2'MOE) ASO exemplifies such an advance. This structure-activity optimized moiety effects receptor-mediated uptake of the ASO prodrug through the asialoglycoprotein receptor 1 to support selective targeting of RNAs expressed by hepatocytes. In this study we report the integrated assessment of data available from randomized placebo-controlled dose-ranging studies of this chemical class of ASOs administered systemically to healthy human volunteers. First, we compare the pharmacokinetic and pharmacodynamic profiles of a subset of the GalNAc 3 -conjugated PS-modified 2'MOE ASOs to the parent PS-modified 2'MOE ASOs for which plasma analytes are available. We then evaluate the safety profile of the full set of GalNAc 3 -conjugated PS-modified 2'MOE ASO conjugates by the incidence of signals in standardized laboratory tests and by the mean laboratory test results as a function of dose level over time. With hepatocyte targeted delivery, the ED 50 for the GalNAc 3 -conjugated PS-modified 2'MOE ASO subset ranges from 4 to 10 mg/week, up to 30-fold more potent than the parent PS-modified 2'MOE ASO. No GalNAc 3 -conjugated PS-modified 2'MOE ASO class effects were identified from the assessment of the integrated laboratory test data across all doses tested with either single or multidose regimens. The increase in potency supports an increase in the safety margin for this new chemical class of ASOs now under broad investigation in the clinic. Although the total exposure is limited in the initial phase 1 trials, ongoing and future investigations in patient populations will support evaluation of the effects of long-term exposure.

Successful immunogene therapy depends not only on the therapeutic gene but also on the gene delivery vector. In this study, we synthesized a novel copolymer consisting of low-molecular-weight polyethylenimine (PEI) cross-linked by myo-inositol (INO) and conjugated with a galactose-grafted PEG chain, named LA-PegPI. We characterized the chemical structure and molecular weight of the copolymer and particle properties of LA-PegPI/pDNA. Furthermore, we showed that LA-PegPI/pDNA polyplexes possessed excellent stability in physiological salt solution, low cytotoxicity, and high transfection efficiency in the asialoglycoprotein receptor (ASGPR)-positive liver cells in vitro. Importantly, we also showed that through intraperitoneal injection of LA-PegPI/pDNA nanoparticles, the reporter gene was forcefully expressed in the liver hepatocytes of mice. Finally, we documented that intraperitoneal injection of LA-PegPI/pIL15 nanoparticles effectively suppressed tumor growth and prolonged survival time of tumor-bearing mice via activation of CD8 + T cells and NK cells and upregulation of the cytokines IFN-γ, TNF, and IL12 in an orthotopic hepatocellular carcinoma mouse model. Interestingly, LA-PegPI/pluc nanoparticles could effectively stimulate the proliferation of NK cells and inhibit tumor growth in this model. In summary, LA-PegPI is a useful gene vector for immunogene therapy of hepatocellular carcinoma, and its potential for clinical application warrants further study., (Copyright © 2018. Published by Elsevier Ltd.)

C-type lectins (CTLs) have a diverse range of functions including cell-cell adhesion, immune response to pathogens and apoptosis. Asialoglycoprotein receptor (ASGPR), also known as hepatic lectin, a member of CTLs, was the first animal lectin identified, yet information regarding it remains rather limited in teleost. In this study, we identified a putative protein in zebrafish, named as the zebrafish hepatic lectin (Zhl). The zhl encoded a typical Ca 2+ -dependent carbohydrate-binding protein, and was mainly expressed in the liver in a tissue specific fashion. Challenge with LPS and LTA resulted in significant up-regulation of zhl expression, suggesting involvement in immune response. Actually, recombinant C-type lectin domain (rCTLD) of Zhl was found to be capable of agglutinating and binding to both Gram-negative and Gram-positive bacteria and enhancing the phagocytosis of the bacteria by macrophages. Moreover, rCTLD specifically bound to insoluble lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), which were inhibited by galactose. Interestingly, Zhl was located in the membrane, and its overexpression could inhibit the production of pre-inflammatory cytokines. Taken together, these results indicate that Zhl has immune activity capable of defending invading pathogens, enriching our understanding of the function of ASGPR/hepatic lectin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hematogenous recurrence is a challenging clinical finding that often leads to fatalities of patients with gastric cancer. Therefore, the identification of specific biomarkers and potential therapeutic target molecules for hematogenous recurrence is required to improve the outcomes of these patients. Here, transcriptome and bioinformatics analyses were conducted to uncover candidate molecules differentially expressed in patients with hematogenous recurrence of gastric cancer. One potential candidate identified was asialoglycoprotein receptor 2 ( ASGR2 ), and siRNA experiments were conducted to determine the effect of manipulating ASGR2 expression has on cell phenotypes. ASGR2 mRNA expression analysis using quantitative real-time reverse-transcription PCR was conducted with stage II/III gastric cancer clinical specimens ( n = 95). Transcript levels were increased in gastric cancer cells as compared with a control nontumorigenic epithelial cell line. Knockdown of ASGR2 decreased the adhesion and migration potential. Thus, although gastric cancer cell-invasive activity was significantly decreased by knockdown, forced expression of ASGR2 promoted invasive activity. Using a mouse hepatic metastasis model, knockdown of ASGR2 resulted in the absence of hepatic metastasis formation. High ASGR2 expression in primary gastric cancer tissues was an independent predictor of shorter disease-free and overall survival. Finally, patients with high ASGR2 expression were more likely to have a high cumulative rate of hematogenous recurrence but not peritoneal or nodal recurrence. Implications: ASGR2 expression is associated with the malignant phenotypes in gastric cancer and represents a specific biomarker of hematogenous recurrences after curative resection for gastric cancer. Mol Cancer Res; 16(9); 1420-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl β-d-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson's Disease. However, before siRNAs can become drugs, they must overcome a billion years of evolutionary defenses designed to keep invading RNAs on the outside cells from getting to the inside of cells. Not surprisingly, significant effort has been placed in developing a wide array of delivery technologies. Foremost of these has been the development of N-acetylgalactosamine (GalNAc) siRNA conjugates for delivery to liver. Tris-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs enter the cytoplasm to induce robust, target selective RNAi responses in vivo. Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, are currently underway by three biotech companies to treat a wide variety of diseases. GalNAc-siRNA conjugates are a simple solution to the siRNA delivery problem for liver hepatocytes and have shown the RNAi (and antisense oligonucleotide) field the path forward for targeting other tissue types.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N - vs O -linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O -glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF -/- /Asgr1 -/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N -acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF -/- /Asgr1 -/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1 -/- mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF., (© 2018 by The American Society of Hematology.)

Recently, some studies have reported nephrotoxicity associated with a certain class of antisense oligonucleotides (ASOs) in humans. One possibility for reducing the potential nephrotoxicity of ASOs is to alter their pharmacokinetics. In this study, we investigated the effect of a ligand conjugation strategy on the renal accumulation of ASOs. We selected two ligands, cholesterol and N-acetylgalactosamine (GalNAc), with the purpose of reducing renal distribution and liver targeting, and then designed a series of cholesterol-GalNAc dual conjugated ASOs. The gene-silencing activity of the cholesterol-GalNAc dual conjugated ASO in the liver was slightly lower than that of a GalNAc-conjugated ASO. On the other hand, the renal distribution of the cholesterol-GalNAc dual conjugated ASO was considerably decreased compared with the GalNAc-conjugated ASO, as we expected. As dual conjugation was successful in reducing the renal distribution of ASO, it should be an effective strategy for reducing the nephrotoxic potential of ASOs.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Combination with chemotherapeutic drug and gene therapy has been proven highly effective in suppressing tumor progression. Hence, an asialoglycoprotein receptor (ASGPR)-targeting nanodrug delivery system based on mesoporous silica (MSN) nanocarrier for co-delivery of sorafenib (SO) and vascular endothelial growth factor (VEGF) targeted siRNA (siVEGF) to hepatocellular carcinoma (HCC) was successfully designed and synthesized. The structure of nanoparticles was characterized by IR, particle size, zeta potential and N2 adsorption-desorption. The nanoparticles were further evaluated for drug release, cellular uptake, transfection, cell cytotoxicity and cell cycle against HepG2 and Huh7 cells. In vitro testing demonstrated that MSN-LA delivery system could not only induce S cell cycle arrest, enhance the cytotoxicity and improve the tumor target of SO and siVEGF, but also enhance the siVEGF transfection efficiency in ASGPR-overexpressing Huh7 cells. Overall, the MSN-LA delivery system can be a promising drug carrier which could further enhance the anti-cancer efficacy of SO and siVEGF via the active targeting property of LA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The gene asialoglycoprotein receptor 1 (ASGR1) encodes a subunit of the asialoglycoprotein receptor. Here we report the generation of a human embryonic stem cell line WAe001-A-6 harbouring homozygous ASGR1 mutations using CRISPR/Cas9. The mutation involves a 37bp deletion, resulting in a frame shift. The homozygous knockout WA01 cell line maintains a normal karyotype, typical stem cell morphology, pluripotency and differentiation potential in vitro., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Advancement of RNAi-based therapeutics depends on effective delivery to the site of protein synthesis. Although intravenously administered, multi-component delivery vehicles have enabled small interfering RNA (siRNA) delivery and progression into clinical development, advances of single-component, systemic siRNA delivery have been challenging. In pre-clinical models, attachment of a triantennary N-acetylgalactosamine (GalNAc) ligand to an siRNA mediates hepatocyte uptake via the asialoglycoprotein receptor enabling RNAi-mediated gene silencing. In this phase 1 study, we assessed translation of this delivery approach by evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of a GalNAc-siRNA conjugate, revusiran, targeting transthyretin (TTR). Subjects received a placebo or ascending doses of revusiran subcutaneously ranging from 1.25-10 mg/kg in the single and 2.5-10 mg/kg in the multiple ascending dose phases. Revusiran was generally well tolerated, with transient, mild to moderate injection site reactions the most common treatment-emergent adverse events. Doses of 2.5-10 mg/kg revusiran elicited a significant reduction of serum TTR versus the placebo (p < 0.01), with mean TTR reductions of approximately 90% observed with multiple dosing. These results demonstrate translation of this novel delivery platform, enabling clinical development of subcutaneously administered GalNAc-siRNAs for liver-based diseases., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The mannose receptor (ManR, Mrc1) and asialoglycoprotein receptor (ASGR, Asgr1 and Asgr2) are highly abundant endocytic receptors expressed by sinusoidal endothelial cells and parenchymal cells in the liver, respectively. We genetically manipulated either receptor individually or in combination, revealing phenotypic changes in female and male mice associated with changes in circulating levels of many glycoproteins. Both receptors rise and fall in response to progesterone during pregnancy. Thirty percent of Asgr2(-/-) and 65% of Mrc1(-/-)Asgr2(-/-) mice are unable to initiate parturition at the end of pregnancy, whereas Mrc1(-/-) mice initiate normally. Twenty five percent of Mrc1(-/-)Asgr2(-/-) male mice develop priapism when mating due to thrombosis of the penile vein, but neither Mrc1(-/-) nor Asgr2(-/-) mice do so. The half-life for luteinizing hormone (LH) clearance increases in Mrc1(-/-) and Mrc1(-/-)Asgr2(-/-) mice but not in Asgr2(-/-) mice; however, LH and testosterone are elevated in all three knockouts. The ManR clears LH thus regulating testosterone production, whereas the ASGR appears to mediate clearance of an unidentified glycoprotein that increases LH levels. More than 40 circulating glycoproteins are elevated >3.0-fold in pregnant Mrc1(-/-)Asgr2(-/-) mice. Pregnancy-specific glycoprotein 23, undetectable in WT mice (<50 ng/ml plasma), reaches levels of 1-10 mg/ml in the plasma of Mrc1(-/-)Asgr2(-/-) and Asgr2(-/-) mice, indicating it is cleared by the ASGR. Elevation of multiple coagulation factors in Mrc1(-/-)Asgr2(-/-) mice may account for priapism seen in males. These male and female phenotypic changes underscore the key roles of the ManR and ASGR in controlling circulating levels of numerous glycoproteins critical for regulating reproductive hormones and blood coagulation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The asialoglycoprotein receptor (ASGR), which is expressed mainly in hepatocytes, is downregulated in poorly differentiated hepatocellular carcinoma (HCC). Here we investigated the role of ASGR1 in HCC metastasis as well as the possible underlying molecular mechanisms. We found that ASGR1 was downregulated in HCC tissue compared with adjacent non-tumorous liver tissue and that lower ASGR1 expression was associated with higher TNM stage and poorer prognosis in HCC patients. ASGR1 overexpression inhibited hepatoma cell migration and invasion in vitro and in vivo, while ASGR1 knockdown had the opposite effects. Furthermore, ASGR1 interacted directly with human longevity assurance homolog 2 of yeast LAG1 (LASS2). Knockdown of LASS2 attenuated the inhibitory effects of ASGR1 on hepatoma cell migration and invasion in vitro. ASGR1 decreased V-ATPase activity in hepatoma cells, and this was reversed by LASS2 knockdown. Finally, HCC patients with low LASS2 levels had poor prognosis, while those with high ASGR1 and LASS2 levels had better prognosis. Thus, ASGR1 may act as a potential metastasis suppressor in HCC, and the combination of ASGR1 and LASS2 may help predict the prognosis of HCC patients., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Several sequence variants are known to have effects on serum levels of non-high-density lipoprotein (HDL) cholesterol that alter the risk of coronary artery disease., Methods: We sequenced the genomes of 2636 Icelanders and found variants that we then imputed into the genomes of approximately 398,000 Icelanders. We tested for association between these imputed variants and non-HDL cholesterol levels in 119,146 samples. We then performed replication testing in two populations of European descent. We assessed the effects of an implicated loss-of-function variant on the risk of coronary artery disease in 42,524 case patients and 249,414 controls from five European ancestry populations. An augmented set of genomes was screened for additional loss-of-function variants in a target gene. We evaluated the effect of an implicated variant on protein stability., Results: We found a rare noncoding 12-base-pair (bp) deletion (del12) in intron 4 of ASGR1, which encodes a subunit of the asialoglycoprotein receptor, a lectin that plays a role in the homeostasis of circulating glycoproteins. The del12 mutation activates a cryptic splice site, leading to a frameshift mutation and a premature stop codon that renders a truncated protein prone to degradation. Heterozygous carriers of the mutation (1 in 120 persons in our study population) had a lower level of non-HDL cholesterol than noncarriers, a difference of 15.3 mg per deciliter (0.40 mmol per liter) (P=1.0×10(-16)), and a lower risk of coronary artery disease (by 34%; 95% confidence interval, 21 to 45; P=4.0×10(-6)). In a larger set of sequenced samples from Icelanders, we found another loss-of-function ASGR1 variant (p.W158X, carried by 1 in 1850 persons) that was also associated with lower levels of non-HDL cholesterol (P=1.8×10(-3))., Conclusions: ASGR1 haploinsufficiency was associated with reduced levels of non-HDL cholesterol and a reduced risk of coronary artery disease. (Funded by the National Institutes of Health and others.).

The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve targeted nanomedicines for a variety of applications.

Background: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury., Methods: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs., Results: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model., Conclusions: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.

Background: Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD, respectively) are major health problems, as patients with either condition can progress to hepatitis, fibrosis, and cirrhosis. Although histologically similar, key differences likely exist in these two models. For example, altered content of several vesicle trafficking proteins have been identified in AFLD, but their content in NAFLD is unknown. In this study, we compared select parameters in NAFLD and AFLD in a rat model., Methods: We fed either Lieber- DeCarli liquid control or alcohol-containing (35 % as calories) diet (AFLD model) or lean or high-fat (12 or 60 % derived from fat, respectively) pellets (NAFLD model) for 8-10 weeks, n = 8 in each model. Serum, hepatocytes and liver tissue were analyzed. Liver injury markers were measured in serum, triglyceride content and endocytosis (binding and internalization of (125)I- asialoorosomucoid) was measured in isolated hepatocytes, and content of selected trafficking proteins (Rab3D, Rab7 and Rab18) were determined in whole liver tissue., Results: Although liver injury markers and triglyceride content were similar in both models, binding and internalization of (125)I- asialoorosomucoid was significantly impaired in the hepatocytes from AFLD, but not NAFLD, animals. In addition, protein content of the asialoglycoprotein receptor (ASGPR) and three trafficking proteins, Rab3D, Rab7and Rab18, were significantly decreased after alcohol, but not high-fat feeding. Levels of protein carbonylation, amount of glutathione stores, and lipid peroxidation were similar irrespective of the insult to the livers that resulted in fatty liver., Conclusion: Impairments in protein trafficking in AFLD are likely a direct result of alcohol administration, and not a function of fatty liver.

Background: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia., Methods: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers., Results: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion., Conclusions: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. Here we demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic expression of thrombopoietin (TPO) mRNA and protein, thereby regulating platelet production. Endocytic AMR controls TPO expression through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this newly identified physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as essential thrombocythemia and immune thrombocytopenia, and contributes to an understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.

The 17p13.1 microdeletion syndrome is a recently described genomic disorder with a core clinical phenotype of intellectual disability, poor to absent speech, dysmorphic features, and a constellation of more variable clinical features, most prominently microcephaly. We identified five subjects with copy-number variants (CNVs) on 17p13.1 for whom we performed detailed clinical and molecular studies. Breakpoint mapping and retrospective analysis of published cases refined the smallest region of overlap (SRO) for microcephaly to a genomic interval containing nine genes. Dissection of this phenotype in zebrafish embryos revealed a complex genetic architecture: dosage perturbation of four genes (ASGR1, ACADVL, DVL2, and GABARAP) impeded neurodevelopment and decreased dosage of the same loci caused a reduced mitotic index in vitro. Moreover, epistatic analyses in vivo showed that dosage perturbations of discrete gene pairings induce microcephaly. Taken together, these studies support a model in which concomitant dosage perturbation of multiple genes within the CNV drive the microcephaly and possibly other neurodevelopmental phenotypes associated with rearrangements in the 17p13.1 SRO., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

This study depicts the 'click' construction of a water-soluble galactosyl rhodamine that can selectively probe mercury ions internalized by hepatoma cells over other cancer cells.