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Excessive complement activation plays a pivotal role in a variety of disorders. Complement component C5 is a clinically validated therapeutic target for treatment of both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome. We developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver produces essentially the entirety of C5 and other complement pathway proteins. We are developing ALN-CC5, an investigational RNAi therapeutic targeting human, primate and rodent C5. C5 silencing and complement activity inhibition were examined in rodents and primates. Multi-dose SC ALN-CC5 treatment resulted in sustained lowering of cyno serum C5 with ≤3% residual protein remaining. C5 reduction was associated with >90% and >95% inhibition of classical and alternative complement pathways as measured by ELISA-based assays. Additionally, >80% lowering of complement serum hemolytic activity was observed. ALN-CC5 was safe and well tolerated in both rat and non-human primate toxicology studies. In addition to wild type animals, ALN-CC5 was tested in several animal models of disease in which complement activation plays a prominent role. Silencing of murine C5 was highly efficacious in a model of anti-collagen antibody-induced arthritis with a disease modifying activity equivalent to that of an anti-C5 antibody. Furthermore, C5 silencing was effective at reducing proteinuria in a rat model of membranous nephropathy. Up-regulation of C5 expression, observed in both models, had no effect on the extent of C5 silencing, suggesting that ALN-CC5 could be efficacious in the context of inflammation. These data demonstrate a prominent role for circulating, liver-derived C5 in mediating pathology at extrahepatic sites and the potential utility of an RNAi therapeutic targeting C5. In summary, RNAi-mediated silencing of liver-derived C5 is a promising novel therapeutic approach for inhibiting systemic complement activity, with the potential to enable, low volume, subcutaneous treatment for patients with PNH and other disorders where complement activation plays a role in disease progression. Disclosures Borodovsky: Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Banda:Alnylam: Research Funding. Holers:Alnylam: Research Funding. Vaishnaw:Alnylam Pharmaceuticals: Employment, Equity Ownership. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Charisse:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Salant:Alnylam: Honoraria. Fitzgerald:Alnylam: Employment. Meyers:Alnylam: Employment. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6 expression significantly ameliorated the disease phenotype in the Hbbth3/+ mouse model of Thalassemia Intermedia (Blood. 2013; 14;121(7):1200-8). The Tmprss6 gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6 removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6 in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+ mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia. To this end, we developed ALN-TMP, a subcutaneous RNAi therapeutic targeting hepatic Tmprss6 for the treatment of β-Thalassemia. ALN-TMP employs the GalNAc conjugate siRNA delivery platform that safely and effectively delivers siRNA to the liver for hepatic gene silencing. Preclinical animal data demonstrate ALN-TMP exhibits robust and durable dose-dependent gene suppression as single dose administration of ALN-TMP leads to > 80% Tmprss6 gene suppression for up to 3 weeks post-dose. This leads to concomitant increases in Hepcidin gene expression (>2x baseline levels) and subsequent decreases in total serum iron and TfSat (>50% decrease from baseline). The degree to which ALN-TMP modulates Hepcidin and serum iron mobilization is nearly identical to that observed in the previous Hbbth3/+ mouse studies and suggests ALN-TMP is a potent RNAi therapeutic with the potential of producing disease modifying effects in β-Thalassemia. Disclosures: Butler: Alnylam: Employment. Fishman:Alnylam: Employment. Racie:Alnylam Pharmaceutical, Inc: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Fitzgerald:Alnylam: Employment.

Abstract 3161 Anemia in chronic kidney disease patients due to impaired renal production of erythropoietin (EPO) and insufficient erythropoiesis is a significant public health problem. One approach to compensate for the low EPO levels in patients with impaired kidney function would be to stimulate the levels of EPO production from non-renal sources. It is well known that during fetal development the liver serves as the primary producer of EPO until the kidney becomes the dominant source after birth. Negative regulation of EPO production is mediated by the EGLN family of prolyl hydroxylases (PHDs1-3) that play an important role in oxygen sensing by targeting the transcription factor hypoxia inducible factor (HIF) for degradation via the proteasome. HIF stabilization and activity is required to mediate EPO gene transcription. Previously it has been shown that liver specific conditional knockout of all three PHDs is required to activate HIF and induce EPO production in liver (Minamishima et al. Science 2010). Here we show that simultaneous siRNA mediated knockdown of all 3 PHD genes in mouse liver using lipid nanoparticles (LNPs) can induce hepatic EPO mRNA activation, elevation of serum EPO levels and stimulation of erythropoiesis. We extend these data by examining siRNA knockdown of different combinations of the 3 PHD genes and demonstrate that PHD2 is the dominant PHD gene regulating hepatic EPO production. In addition, due to the specificity of the LNP delivery system we show that the HIF activation and increase in EPO mRNA occurs specifically in liver. Increases in serum EPO and hematocrit were durable for two weeks and one month after a single intravenous dose of LNP siRNA. Furthermore, PHD siRNA silencing in a 5/6 nephrectomy model successfully elevates hemoglobin levels and corrects anemia. In conclusion, targeting of EGLN prolyl hydroxylase genes with siRNA therapeutics has potential in the treatment of anemia associated with chronic kidney disease and provides liver-specific target gene regulation. Disclosures: Querbes: Alnylam Pharmaceuticals: Employment. Bogorad:Alnylam Pharmaceuticals: Research Funding. Moslehi:Alnylam Pharmaceuticals: Honoraria. Akinc:Alnylam Pharmaceuticals: Employment. Wong:Alnylam Pharmaceuticals: Employment. Zurenko:Alnylam Pharmaceuticals: Employment. Qin:Alnylam Pharmaceuticals: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Kuchimanchi:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Fitzgerald:Alnylam Pharmaceuticals: Employment. Kotelianski:Alnylam Pharmaceuticals: Employment. Kaelin:Fibrogen: Consultancy, Equity Ownership.