MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, the level of which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding mRNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of β-catenin signaling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumor angiogenesis. Overexpression of miR-9 in otherwise-non-metastatic breast tumor cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 using a ‘miRNA sponge’ in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumor grade, and metastatic status. These findings uncover a regulatory and signaling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.

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Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

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ALN-AT3, a subcutaneously administered RNAi therapeutic targeting antithrombin (AT), is currently being developed for the treatment of hemophilia and rare bleeding disorders. It has previously been demonstrated that once weekly dosing of ALN-AT3 results in potent, dose-dependent and reversible silencing of plasma AT in multiple preclinical species. In the mouse, steady state ED50 knockdown was achieved following weekly doses of 0.5 mg/kg. The objective of this study was to evaluate and compare the tolerability (exaggerated pharmacology) of ALN-AT3 when administered weekly to wild-type (WT) versus hemophilia A (HA) mice. Methods ALN-AT3, diluted in PBS, was administered once weekly (x 7 weeks) via subcutaneous injection at 0, 10, 30, and 100 (HA only) mg/kg. The dose levels used in this study represented 20-, 60- and 200-fold dose multiples of the mouse ED50. Five animals per group were to be terminated on Days 23 and 44 for post-mortem evaluation. Potential test article-related effects were evaluated by clinical signs, body weight, clinical pathology (hematology, coagulation), macroscopic observations at necropsy, organ weights, and histopathology of select tissues (eye, heart, kidney, liver, lung, spleen, gross lesions). The pharmacodynamic effects of ALN-AT3 were evaluated by plasma AT protein assay. Results Repeat administration of ≥ 10 mg/kg ALN-AT3 to WT mice was not tolerated, as evidenced by early mortality (8/10 animals at 10 mg/kg, 9/10 animals at 30 mg/kg), adverse clinical signs (ocular abnormalities, craniofacial swelling, lethargy), weight loss, clinical pathology alterations (decreased PLT, increased NEU), increased relative spleen weights, and microscopic findings in heart (thrombi) and eye (hemorrhage). Residual plasma AT protein in WT and HA animals was <10% relative to control (> 90% on-target AT suppression). All findings were suggestive of thrombosis and disseminated intravascular coagulation, consistent with the intended pro-coagulant effect of AT suppression. The findings in WT animals are also consistent with heterozygous AT deficiency in humans, which is almost always lethal in utero. In contrast, repeat administration of ALN-AT3 was well tolerated in HA mice, with no adverse findings up to 100 mg/kg. In addition, HA mice exhibited significant reductions in ex vivo clotting times (aPTT) to values comparable to control WT animals, consistent with the therapeutic hypothesis of rebalancing the hemostatic system in the disease condition. Collectively, the data also suggest an expanded therapeutic index of AT suppression in the hemophilia disease condition. Disclosures: Barros: Alnylam Pharmaceuticals: Employment. Carioto:Alnylam Pharmaceuticals: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Jiang:Alnylam Pharmaceuticals: Employment. Qin:Alnylam Pharmaceuticals: Employment. Prabhala:Alnylam Pharmaceuticals: Employment. Sehgal:Alnylam Pharmaceuticals: Employment. Akinc:Alnylam Pharmaceuticals: Employment.