Conjugation of small interfering RNA (siRNA) with lipophilic molecules has been demonstrated to enhance cellular uptake in cell culture and to produce efficient endogenous gene silencing in the liver ...after systemic administration and in neurons after direct local injection. Here, we evaluated the
in vivo delivery of siRNAs conjugated with different linkers to cholesterol by targeting CNPase (2′-3′-cyclic nucleotide 3′-phosphodiesterase) in oligodendrocytes. Cholesterol-conjugated siRNAs administered to the rat corpus callosum by intraparenchymal central nervous system (CNS) infusion show improved silencing ability compared with unconjugated siRNA. Furthermore, conjugation of siRNA to cholesterol with a cleavable disulfide linker appears to be beneficial for improving the potency of silencing of CNPase mRNA in oligodendrocytes
in vivo. Taken together, these findings indicate that cholesterol-conjugated siRNAs are effective for direct CNS delivery to oligodendrocytes, and that the biocleavable disulfide linker appears to be beneficial for improving the potency of silencing of target mRNA
in vivo.
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Acute hepatic porphyria (AHP) is a family of rare metabolic disorders including acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP), caused by a ...deficiency in one of the eight enzymes required for heme biosynthesis in the liver. When ALA synthetase (ALAS1), the first and rate limiting step in the pathway, is induced by triggers such as exposure to certain drugs or fasting, the neurotoxic heme intermediates aminolevulinic acid (ALA) and porphobilinogen (PBG) can accumulate upstream of the deficient enzyme leading to acute and potentially life threatening neurovisceral attacks in AHP patients.
RNA interference is a naturally occurring cellular mechanism mediated by small interfering RNA (siRNA) that allows for the inhibition of protein synthesis through the cleavage and degradation of a specific mRNA. ALN-AS1 is an investigational RNAi therapeutic that targets ALAS1 in order to decrease ALA and PBG levels and subsequent porphyria attacks. We are currently conducting a phase 1, multinational, randomized, placebo-controlled, study in 3 parts; Part A single ascending dose (SAD), Part B multiple ascending dose (MAD) and Part C multiple dose (MD) study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics (i.e. changes in ALA, PBG and circulating ALAS1 mRNA levels) of subcutaneously administered ALN-AS1 in AIP patients. Part C also has exploratory analyses of clinical activity, including the impact of ALN-AS1 on porphyria attacks, acute healthcare visits, and heme treatment (ClinicalTrials.gov Identifier: NCT02452372).
We previously presented interim SAD data demonstrating that ALN-AS1 was generally well tolerated with no serious adverse events (SAEs) or clinically significant laboratory abnormalities related to study drug, and no discontinuations due to AEs. Circulating ALAS1 mRNA levels had a mean (SEM) maximal reduction of 44% ± 8% relative to baseline (p ≤ 0.01 compared to placebo), with concomitant mean (SEM) maximal reductions in ALA and PBG of 77% ± 7% and 73% ± 6%, respectively (p= 0.03 and 0.06 compared to placebo, respectively) at the 0.35 mg/kg dose. In addition, changes in circulating ALAS1 mRNA were highly correlated with changes in urinary ALA and PBG (R2=0.82, p<10-15). We will now report interim progress and data from all 3 Parts of the phase 1 study including safety, pharmacodynamics, pharmacokinetics and exploratory clinical activity.
Sardh:Alnylam Pharmaceuticals: Consultancy. Harper:Alnylam Pharmaceuticals: Consultancy. Balwani:ICGG Gaucher Registry (Sanofi Genzyme sponsored): Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Consultancy. Anderson:Alnylam Pharmaceuticals: Consultancy. Bloomer:Alnylam Pharamceuticals: Consultancy. Bissel:Alnylam Pharmaceuticals: Consultancy. Desnick:Alnylam Pharmaceuticals: Consultancy. Bonkovsky:Alnylam Pharamceuticals: Consultancy. Penz:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chan:Alnylam Pharmaceuticals: Employment, Equity Ownership. Soh:Alnylam Pharmaceuticals: Employment, Equity Ownership. Querbes:Alnylam Pharmaceuticals: Employment, Equity Ownership. Simon:Alnylam Pharmaceuticals: Employment, Equity Ownership. Rees:Alnylam Pharmaceuticals: Consultancy.
The cover picture shows recognition of siRNA conjugated to trivalent N-acetylgalactosamine by the asialoglycoprotein receptor (ASGPR) expressed on the surface of a hepatocyte, and intracellular ...events that result in gene silencing. In nature, ASGPR mediates clearance of "dead" glycoproteins from circulation. This natural molecular machinery, which recognizes exposed galactoses, has been exploited to deliver nucleic acid-based drugs into hepatocytes. Engineered GalNAc moieties in favorable proximity and optimal spatial orientation serve as ligand mimics and are recognized by ASGPR leading to cargo internalization. GalNAc monomers were synthesized by using non-nucleosidic tethers and incorporated via phosphodiester linkages into oligonucleotides. The optimal siRNA-GalNAc conjugate was prepared by sequential covalent incorporation of three single GalNAc moieties at the 3'-end of the sense strand of siRNA. The siRNA-GalNAc conjugate elicited robust gene silencing in liver. Clinical experience with siRNA-GalNAc indicates that this strategy is a breakthrough in the tissue-specific delivery of therapeutic nucleic acids. For more details see the communication by K. G. Rajeev, M. Manoharan et al. on
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.
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.
The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies ...describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.
The common human polyomavirus JCV establishes persistent infection in nearly 30-70 percent of the world's population. In immunosuppressed individuals, such as AIDS patients, the virus can spread from ...sites of latency to the brain resulting in the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). A better understanding of the basic biology of the virus life cycle is required to identify potential sites of therapeutic intervention. Here we utilize a number of approaches including confocal microscopy, RNAi, and dominant negative mutants of key proteins involved in endocytosis and cellular trafficking to investigate mechanisms of JCV cellular invasion. We show that JCV enters cells by a ligand inducible clathrin mediated mechanism dependent on tyrosine kinases and the clathrin associated protein Epidermal growth factor substrate clone 15 (eps15). Upon binding to host cells JCV activates signaling events that lead to downstream activation of the MAP Kinases ERK1 and ERK2. Cell fractionation, detergent wash-out experiments, and single cell infection assays indicated that JCV proceeds from clathrin coated pits to cavaelae docked on early endosomes and this process is dependent on the Rab5 GTPase and cholesterol. Finally, JCV infection but not cellular entry is inhibited by expression of shRNA to caveolin-1. Co-localization studies with fluorescently labeled virus suggest JCV is further transported in caveolae to caveosomes and then to the Endoplasmic Reticulum. We conclude by defining a novel transport pathway from early endosomes to caveosomes exploited by JCV and have identified a novel role for caveolae in the endosomal sorting of non-caveolar ligands.
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