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.

A Drug-Drug Interaction Study Evaluating the Effect of Givosiran, a Small Interfering Ribonucleic Acid, on Cytochrome P450 Activity in the Liver.

Givosiran (trade name GIVLAARI) is a small interfering ribonucleic acid that targets hepatic delta-aminolevulinic acid synthase 1 (ALAS1) messenger RNA for degradation through RNA interference (RNAi) that has been approved for the treatment of acute hepatic porphyria (AHP). RNAi therapeutics, such as givosiran, have a low liability for drug-drug interactions (DDIs) because they are not metabolized by cytochrome 450 (CYP) enzymes, and do not directly inhibit or induce CYP enzymes in the liver. The pharmacodynamic effect of givosiran (lowering of hepatic ALAS1, the first and rate limiting enzyme in the heme biosynthesis pathway) presents a unique scenario where givosiran could potentially impact heme-dependent activities in the liver, such as CYP enzyme activity. This study assessed the impact of givosiran on the pharmacokinetics of substrates of 5 major CYP450 enzymes in subjects with acute intermittent porphyria (AIP), the most common type of AHP, by using the validated "Inje cocktail," comprised of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). We show that givosiran treatment had a differential inhibitory effect on CYP450 enzymes in the liver, resulting in a moderate reduction in activity of CYP1A2 and CYP2D6, a minor effect on CYP3A4 and CYP2C19, and a similar weak effect on CYP2C9. To date, this is the first study evaluating the DDI for an oligonucleotide therapeutic and highlights an atypical drug interaction due to the pharmacological effect of givosiran. The results of this study suggest that givosiran does not have a large effect on heme-dependent CYP enzyme activity in the liver.

[New therapeutic option for acute hepatic porphyrias]. / Neue Therapieoption für akute hepatische Porphyrien.

Givosiran is a small synthetic double-stranded siRNA (small interfering RNA) conjugated with N-acetyl-galactosamine (GalNAc) for specific hepatocyte targeting via the asialoglycoprotein receptor. A prospective randomized multicenter study (Envision) demonstrated the clinical efficacy of monthly subcutaneous injection of Givosiran for the prevention of attacks of acute hepatic porphyria (AHP). This leads to highly selective transcriptional inhibition of the key hepatic enzyme, aminolaevulinate synthase 1, that is overexpressed in AHP. The success of the Envision study has led to the approval of Givosiran in the US and Europe for the treatment of severe AHP. This innovative guided siRNA therapy has opened up the possibility to selectively inhibit the expression of any hepatocyte gene whose overexpression that causes pathology, which can be considered a milestone development in hepatology. However, currently this treatment with givosiran is very costly. Moreover, since some patients experience worsening of kidney function and elevated aminotransferases, monthly monitoring of these parameters is necessary in the first half year of treatment.

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.

Nonclinical Pharmacokinetics and Absorption, Distribution, Metabolism, and Excretion of Givosiran, the First Approved N-Acetylgalactosamine-Conjugated RNA Interference Therapeutic.

Givosiran is an N-acetylgalactosamine-conjugated RNA interference therapeutic that targets 5'-aminolevulinate synthase 1 mRNA in the liver and is currently marketed for the treatment of acute hepatic porphyria. Herein, nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous administration with relatively short plasma elimination half-life (t1/2; less than 4 hours). Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor-mediated uptake, and the t1/2 in the liver was significantly longer (∼1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of one nucleotide from the 3' end of antisense strand, AS(N-1)3' givosiran, which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran shows similar pharmacokinetics and ADME properties across rats and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in adequate exposure of givosiran to the target organ (liver). These studies support the interpretation of toxicology studies, help characterize the disposition of givosiran in humans, and support the clinical use of givosiran for the treatment of acute hepatic porphyria.

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.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

The Evolution of Hemophilia Care: Clinical and Laboratory Advances, Opportunities, and Challenges.

Hemophilia A (HA) and B (HB) are X-linked bleeding disorders caused by mutations in the F8 or F9 gene that result in the absence, or reduced activity, of the corresponding clotting factor. The severity of bleeding and related complications is proportional to the amount of residual circulating functional factor. The development of a safe and effective hemophilia treatment lasted several decades and has been mainly based on clotting factor replacement. Advances in the engineering and manufacturing of clotting concentrates have led to the widespread availability of extended half-life products that reduced the number of intravenous infusions needed to achieve adequate trough levels. The recent development of new nonfactor replacement treatments and biotechnology techniques has offered therapeutic alternatives for hemophilia patients with and without inhibitors. These are characterized by an easier route of administration, low immunogenicity, and, regarding gene therapy and cell-based treatments, potential long-term protection from bleeding after a single treatment course. In this review, we analyze recent progresses in the management of hemophilia and discuss opportunities and challenges.

Deficiency of O-linked-glycosylation regulates activation of T cells and aggravates Concanavalin A-induced liver injury.

OBJECTIVE: Protein glycosylation is involved in immunological recognition and immune cell activation. The role of O-glycosylation in Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) was elucidated in the present study. METHODS: Mice were intravenously injected with Con A (10 mg/kg) to establish an AIH mouse model. Here, 24 h prior to administration of Con A, experimental mice were intragastrically administrated with O-glycosylation inhibitor (benzyl-α-GalNAc) at doses of 1 and 5 mg/kg, respectively, while control mice were administrated with the same volume of saline. Before and after administration of Con A for 6 and 12 h, mice were sacrificed and their plasma and livers were collected to score liver injury. Peripheral blood, spleen, and thymus were collected for flow cytometry analysis. The expression levels of neutrophilic alkaline phosphatase-3 (NALP3) and NALP6 in liver were evaluated as well. RESULTS: Pre-treatment with benzyl-α-GalNAc increased the serum transaminase levels and induced more infiltration and necrosis in livers of Con A administrated mice. The levels of some pro-inflammation cytokines also increased in administrated mice. In addition, pretreatment with benzyl-α-GalNAc up-regulated the expression levels of NALP3 and NALP6. And benzyl-α-GalNAc inhibited the levels of apoptosis of thymus cells and influenced activation of T cells in peripheral blood and spleen of Con A administrated mice, especially that accelerated the physiological progression of CD4+CD25-CD69+ subset. CONCLUSION: The present research demonstrated that benzyl-α-GalNAc aggravated Con A-induced AIH, and the role of the O-glycosylation inhibitor as the aggravation may be related to regulation of the levels of cytokines, as well as influencing proliferation of T cells.

Givosiran: First Approval.

Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. Givosiran is being developed by Alnylam Pharmaceuticals for the treatment of acute hepatic porphyria (AHP). In November 2019, givosiran was approved in the USA for the treatment of adults with AHP based on the positive results from the multinational, phase III ENVISION trial. In the EU, givosiran received a positive opinion in January 2020 for the treatment of AHP in adults and adolescents aged 12 years and older. This article summarizes the milestones in the development of givosiran leading to this first approval for the treatment of adults with AHP.

Pharmacokinetics and Pharmacodynamics of the Small Interfering Ribonucleic Acid, Givosiran, in Patients With Acute Hepatic Porphyria.

Givosiran is a small interfering ribonucleic acid agent that was recently approved in the United States for the treatment of acute hepatic porphyria (AHP). This phase I study evaluated the safety, pharmacokinetic, and pharmacodynamic profile of subcutaneously (SC) administered givosiran in patients with acute intermittent porphyria, the most common AHP type. Givosiran was rapidly absorbed from the SC injection site with peak plasma concentrations achieved within 0.5-5 hours followed by elimination with a short half-life of 4-10 hours. Plasma exposures of AS(N-1)3' givosiran, an active metabolite with equal potency as givosiran, was 35%-75%. Givosiran treatment resulted in a rapid and dose-dependent reduction in urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) towards the upper limit of normal (ULN) in AHP patients. Greater and more sustained reductions in ALA and PBG were achieved with once monthly dosing compared with once quarterly dosing. After monthly dosing, trough ALA levels were reduced to below the ULN, approximately 95% reduction from baseline, at both the 2.5 and 5.0 mg/kg doses.

Leading RNA Interference Therapeutics Part 2: Silencing Delta-Aminolevulinic Acid Synthase 1, with a Focus on Givosiran.

In November 2019 givosiran became the second small interfering RNA (siRNA)-based drug to receive US Food and Drug Administration (FDA) approval, it has been developed for the treatment of acute intermittent porphyria (AIP), a disorder characterized by life-threatening acute neurovisceral attacks. The porphyrias are a group of disorders in which enzymatic deficiencies in heme production lead to toxic accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG), which are involved in the neurovisceral attacks. Givosiran acts as a conventional siRNA to trigger RNA interference (RNAi)-mediated gene silencing on delta-ALA synthase 1 (ALAS1), thus returning ALA and PBG metabolites to the physiological level to attenuate further neurotoxicity. Givosiran makes use of a new hepatic-delivery system that conjugates three GalNac (N-acetylgalactosamine) molecules to the siRNA passenger strand. GalNac binds to the liver asialoglycoprotein receptor, favoring the internalization of these GalNac-conjugated siRNAs into the hepatic cells. In a phase I study, subcutaneous monthly administration of givosiran 2.5 mg/kg reduced > 90% of ALA and PBG content. This siRNA is being analyzed in ENVISION (NCT03338816), a phase III, multicenter, placebo-controlled randomized controlled trial. In preliminary results, givosiran achieved clinical endpoints for AIP, reducing urinary ALA levels, and presented a safety profile that enabled further drug development. The clinical performance of givosiran revealed that suppression of ALAS1 by GalNac-decorated siRNAs represents an additional approach for the treatment of patients with AIP that manifests recurrent acute neurovisceral attacks.

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.

Integrated Assessment of the Clinical Performance of GalNAc3-Conjugated 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides: I. Human Volunteer Experience.

Advances in medicinal chemistry have produced new chemical classes of antisense oligonucleotides (ASOs) with enhanced therapeutic properties. Conjugation of the triantennary N-acetylgalactosamine (GalNAc3) 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 GalNAc3-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 GalNAc3-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 ED50 for the GalNAc3-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 GalNAc3-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.

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.

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus.

Lipid nanoparticles (LNPs) are the leading technology for delivering short interfering RNA (siRNA) in vivo. While numerous attempts to improve the efficiency of siRNA delivery have been reported, only a few studies of the mechanism of LNP-mediated toxicity and attempts to develop safe LNPs in vivo have been reported, in spite of the significance of such systems, in the light of treatment and clinical applications. We herein report on the elucidation of the mechanism of hepatotoxicity following the intravenous injection of a high dose of hepatotropic LNPs. The LNPs accumulated in liver sinusoidal endothelial cells (LSECs), resulting in their activation and the induction of several cytokines related to neutrophils, followed by neutrophilic inflammation. To circumvent this toxic cascade, the LNPs were modified with a hepatocyte-specific ligand, N-acetyl-d-galactosamine (GalNAc), which resulted in a substantial improvement of hepatocyte-specificity and in a dramatic reduction in toxicity. Moreover, modification of the GalNAc-LNPs with polyethyleneglycol abrogated the LNP-associated toxicity without any detectable loss of gene silencing activity in hepatocytes. Finally, we observed that a single injection of the LNPs resulted in a significant reduction of hepatitis B virus (HBV) genomic DNA and their antigens without any sign of toxicity in chimeric mice with humanized livers that had been persistently infected with HBV. These lines of the fact suggest that the newly designed siRNA-loaded LNPs promise to be a useful technology for the treatment of liver diseases.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

A Highly Durable RNAi Therapeutic Inhibitor of PCSK9.

BACKGROUND: Inclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. METHODS: In this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. RESULTS: The most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84). CONCLUSIONS: In this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442 .).

Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes.

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.