GalNAc-siRNA conjugates: Prospective tools on the frontier of anti-viral therapeutics.

The growing use of short-interfering RNA (siRNA)-based therapeutics for viral diseases reflects the most recent innovations in anti-viral vaccines and drugs. These drugs play crucial roles in the fight against many hitherto incurable diseases, the causes, pathophysiologies, and molecular processes of which remain unknown. Targeted liver drug delivery systems are in clinical trials. The receptor-mediated endocytosis approach involving the abundant asialoglycoprotein receptors (ASGPRs) on the surfaces of liver cells show great promise. We here review N-acetylgalactosamine (GalNAc)-siRNA conjugates that treat viral diseases such as hepatitis B infection, but we also mention that novel, native conjugate-based, targeted siRNA anti-viral drugs may also cure several life-threatening diseases such as hemorrhagic cystitis, multifocal leukoencephalopathy, and severe acute respiratory syndrome caused by coronaviruses and human herpes virus.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of ß-thalassaemia.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

Current and emerging biologics for the treatment of hemophilia.

INTRODUCTION: The development of new biologic agents able to restore thrombin generation has become the focus of innovation in hemophilia management. There is growing interest in the proposal of novel, non-replacement therapy with alternative mechanisms of action and route of administration, hoping to solve still unmet needs in treatment of hemophilic patients with or without inhibitors. AREAS COVERED: The review describes the new molecules, in particular the bi-specific antibody mimicking the coagulation function of FVIII and/or those which work by inhibiting the natural anticoagulants, their mechanism of action and the results of ongoing clinical trials. EXPERT OPINION: Exciting results in enhancing the protection against bleeding and improving quality of life are emerging from clinical trials. However, these molecules with their mechanisms of action also open new problems. Treatment of bleeding and management of surgery in subjects with a rebalanced hemostasis may be difficult, especially for the lack of laboratory tests perfectly reflecting the in vivo coagulation status. A careful surveillance is required to evaluate the risk of thrombotic complication in patients with rebalanced hemostasis, in addition to understand whether these new products offer the same protection on joints as regular prophylaxis with the missing clotting factors.

N-Acetyl-d-galactosamine prevents soya bean agglutinin-induced intestinal barrier dysfunction in intestinal porcine epithelial cells.

Soya bean agglutinin (SBA) is a glycoprotein and the main anti-nutritional component in most soya bean feedstuffs. It is mainly a non-fibre carbohydrate-based protein and represents about 10% of soya bean-based anti-nutritional effects. In this study, we sought to determine the effects of N-Acetyl-D-galactosamine (GalNAc or D-GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC-J2) cells. The IPEC-J2 cells were pre-cultured with 0, 0.125 × 10-4 , 0.25 × 10-4 , 0.5 × 10-4 , 1.0 × 10-4 and 2.0 × 10-4  mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre-incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans-epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre-treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin-3 were lower in the SBA-treated groups without pre-treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin-3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre-treated groups, occludin and claudin-3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC-J2s.

GalNAc bio-functionalization of nanoparticles assembled by electrostatic interactions improves siRNA targeting to the liver.

RNA interference (RNAi) has the potential to reversibly silence any gene with high efficiency and specificity. To fulfill the clinical potential of RNAi, delivery vehicles are required to transport the short interfering RNA (siRNA) to the site of action in the cells of target tissues. Here, we describe the features of novel liver-targeted siRNA nanoparticles (NPs), co-assembled due to the complexation of alginate sulfate (AlgS) with siRNA, mediated by calcium ions bridges (AlgS-Ca2+-siRNA NPs) and then bioconjugation of a targeting ligand onto the AlgS upon the NP surface. To gain insight into the complexation process and confirm AlgS accessibility on NP surface, we investigated different schemes for fabrication. All resulting NPs, independently of the component addition order, were of average size of 130-150nm, had surface charge of <-10mV, exhibited a similar atomic composition on their surface, were efficiently uptaken by HepG2 cells and induced approx. ~90% silencing of STAT3 gene. Ca2+ and AlgS concentrations in NPs affected cell uptake and gene silencing. Bioconjugation of N-acetylgalactosamine (GalNAc), a ligand to the asialoglycoprotein receptor (ASGPR) overexpressed on hepatocytes, was validated by XPS analysis and cell uptake by receptor-mediated mechanism. After intravenous (i.v.) injection to BALB/c mice, GalNAc-NPs were targeted to liver by a factor of ~3 with lesser renal clearance compared to non-targeted NPs. We foresee that the combined advantages of site-specific targeting and reversibility of the tri-component NPs as well as the simplicity of their fabrication make them an attractive system for targeted delivery of siRNA.