Amy Chan

Multivalent N‑Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Treatment of erythropoietin deficiency in mice with systemically administered siRNA

Anemia linked to a relative deficiency of renal erythropoietin production is a significant cause of morbidity and medical expenditures in the developed world. Recombinant erythropoietin is expensive and has been linked to excess cardiovascular events. Moreover, some patients become refractory to erythropoietin because of increased production of factors such as hepcidin. During fetal life, the liver, rather than the kidney, is the major source of erythropoietin. In the present study, we show that it is feasible to reactivate hepatic erythropoietin production and suppress hepcidin levels using systemically delivered siRNAs targeting the EglN prolyl hydroxylases specifically in the liver, leading to improved RBC production in models of anemia caused by either renal insufficiency or chronic inflammation with enhanced hepcidin production.

Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis

Abstract ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression

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.

Sinusoidal and pericellular fibrosis in adult post-transplant liver biopsies: Association with histological changes, hepatic stellate cell activation and impact on patient outcome

Introduction Although the accumulation of hepatic iron occurs during chronic liver injury, its role in fibrogenesis remains poorly understood. To date, studies show that excess iron in hepatocytes promotes formation of reactive oxygen species, which then activate hepatic stellate cells (HSC) to secrete collagen matrix. It is unclear however, whether iron directly affects HSC function. Aim Our aims were to investigate the effects of exogenous iron on core fibrogenic and iron-related genes and proteins in HSCs. Method The GRX murine HSC cell line was treated with the physiologically relevant form of transferrin-bound iron, holotransferrin (0.005, 0.05, 0.5, 2 and 5 g/L) for 24 h, with or without the iron chelator deferoxamine (10 μM). Expression of fibrogenic markers (α-SMA, TGF-β and Serpine-1) were analyzed by qRT-PCR, while ferritin was measured by ELISA. Levels of transferrin receptor (TfR1), TGF-β receptors and p-Smad 2 and 4 were analyzed by western blot, and secreted collagen was measured using the Sircol assay. Results HSCs express the iron-uptake and iron-exporter proteins TfR1 and ferroportin, respectively. Treatment with holotransferrin upregulated the expression of TfR1 by 1.8-fold (p<0.05), led to the accumulation of storage iron (ferritin) by up to 2-fold (p<0.01), and activated HSCs: α-SMA mRNA increased by up to 2-fold (p<0.03), TGF-β mRNA was elevated by 1.6-fold (p=0.05) and Serpine-1 mRNA was raised by 2.5-fold (p<0.05). These were accompanied by a 2-fold increase in secreted collagen (p<0.03), and activation of the TGF-β pathway: increased protein expression of TGF-β R1, TGF-b RII, and p-Smad 2 and 4 (p<0.05). Conversely, the addition of deferoxamine significantly lowered ferritin levels by 30% (p<0.03), repressed fibrogenic genes, α-SMA (0.2-fold; p<0.03), and TGF-β (0.4-fold; <0.03), and reduced collagen secretion by up to 60% (p<0.01). Deferoxamine also inhibited TGF-β signaling, with decreased levels of TGF-β RII and p-Smad 2. Conclusion HSCs express the iron transport proteins and are regulated by levels of exogenous iron. Excess iron activates HSCs and the TGF-β pathway, while iron depletion using deferoxamine attenuates the fibrogenic response, suggesting that iron depletion could be a useful adjunctive therapy in the treatment of individuals with advanced liver disease.