Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing.

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

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.

RNAi-mediated reduction of hepatic Tmprss6 diminishes anemia and secondary iron overload in a splenectomized mouse model of ß-thalassemia intermedia.

Diminished ß-globin synthesis in ß-thalassemia is associated with ineffective erythropoiesis, leading to secondary iron overload caused by inappropriately low levels of hepcidin and to splenomegaly in the symptomatic thalassemias. Splenectomy is often employed in patients with ß-thalassemia to reduce hemolysis. Expression of the iron regulatory peptide hormone hepcidin is repressed by the serine protease TMPRSS6. Hepcidin induction by RNAi-mediated inhibition of TMPRSS6 expression reduces iron overload and mitigates anemia in murine models of ß-thalassemia intermedia. To interrogate the efficacy of RNAi-mediated reduction of Tmprss6 in splenectomized ß-thalassemia, splenectomized ß-thalassemic Hbbth3/+ animals were treated with a GalNAc-conjugated siRNA targeting Tmprss6 (GalNAc-Tmprss6) and their hematological and iron parameters monitored. We demonstrate that treatment with GalNAc-Tmprss6 significantly diminishes Tmprss6 expression and appropriately elevates hepcidin expression in splenectomized Hbbth3/+ animals. Similar to unsplenectomized animals, treated animals have markedly improved anemia due to diminished ineffective erythropoiesis and reduced iron loading in both serum and tissue. These results suggest that RNAi-mediated reduction of Tmprss6 may have positive outcomes even in splenectomized ß-thalassemia patients.

An RNAi therapeutic targeting Tmprss6 decreases iron overload in Hfe(-/-) mice and ameliorates anemia and iron overload in murine ß-thalassemia intermedia.

Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. -thalassemia is a congenital anemia caused by partial or complete loss of -globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe(-/-)) and -thalassemia intermedia (Hbb(th3/+)) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe(-/-) and Hbb(th3/+) mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbb(th3/+) mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of -thalassemia intermedia.

Combination therapy with a Tmprss6 RNAi-therapeutic and the oral iron chelator deferiprone additively diminishes secondary iron overload in a mouse model of ß-thalassemia intermedia.

ß-thalassemias result from diminished ß-globin synthesis and are associated with ineffective erythropoiesis and secondary iron overload caused by inappropriately low levels of the iron regulatory hormone hepcidin. The serine protease TMPRSS6 attenuates hepcidin production in response to iron stores. Hepcidin induction reduces iron overload and mitigates anemia in murine models of ß-thalassemia intermedia. To further interrogate the efficacy of an RNAi-therapeutic downregulating Tmprss6, ß-thalassemic Hbb(th3/+) animals on an iron replete, an iron deficient, or an iron replete diet also containing the iron chelator deferiprone were treated with Tmprss6 siRNA. We demonstrate that the total body iron burden is markedly improved in Hbb(th3/+) animals treated with siRNA and chelated with oral deferiprone, representing a significant improvement compared to either compound alone. These data indicate that siRNA suppression of Tmprss6, in conjunction with oral iron chelation therapy, may prove superior for treatment of anemia and secondary iron loading seen in ß-thalassemia intermedia.

Harnessing a physiologic mechanism for siRNA delivery with mimetic lipoprotein particles.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip-mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex.

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

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.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

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.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.