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Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3'-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2',4'-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2'-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle. Here, we describe the design and characterization of a cEt gapmer DMPK ASO (ISIS 486178), with potent activity in vitro and in vivo against mouse, monkey, and human DMPK. Systemic delivery of unformulated ISIS 486718 to wild-type mice decreased DMPK mRNA levels by up to 90% in liver and skeletal muscle. Similarly, treatment of either human DMPK transgenic mice or cynomolgus monkeys with ISIS 486178 led to up to 70% inhibition of DMPK in multiple skeletal muscles and ∼50% in cardiac muscle in both species. Importantly, inhibition of DMPK was well tolerated and was not associated with any skeletal muscle or cardiac toxicity. Also interesting was the demonstration that the inhibition of DMPK mRNA levels in muscle was maintained for up to 16 and 13 weeks post-treatment in mice and monkeys, respectively. These results demonstrate that cEt-modified ASOs show potent activity in skeletal muscle, and that this attractive therapeutic approach warrants further clinical investigation to inhibit the gain-of-function toxic RNA underlying the pathogenesis of DM1., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443-mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Adhesion molecules are known to be an important part of leukocyte migration and extravasation in both homeostatic and inflammatory conditions. Intracellular adhesion molecule-1 (ICAM-1 or CD54) is constitutively expressed on endothelial cells and is up-regulated during acute and chronic inflammation. We investigated the efficacy and consequences of interfering with CD54 after administration of an antisense oligonucleotide to ICAM-1 (CD54) in the transgenic HLA-B27/beta2 microglobulin rat model. One hundred percent of the HLA-B27 transgene + animals will spontaneously develop chronic inflammation (some more severely than others) in the gastric mucosa, cecum, and colon. We carried out two studies, i.p. injection and rectal administration of antisense. Following i.p. and rectal treatment, there were significant decreases in colonic mucosal wall thickness, histologic inflammation, CD54 expression in the colon and peripheral blood, and the percentage of colon weight per end body weight. Furthermore, decreased expression of CD49d, CD18, and tumor necrosis factor-alpha was observed in antisense treated rats. Therefore, the HLA-B27 transgenic model of spontaneous and chronic inflammatory bowel disease, which has increased expression of adhesion molecules, responds to both routes of administration of ICAM-1 antisense oligonucleotides. These studies support the regulatory role of adhesion molecules in chronic intestinal inflammation, the need for an understanding of how the route of drug delivery can alter the dose and area affected, and finally the role of antisense oligonucleotides as a therapeutic modality in chronic spontaneous inflammatory bowel diseases.

The anti-inflammatory activity of free and liposome-encapsulated oligonucleotide targeted against intercellular adhesion molecule-1 mRNA was investigated in a delayed type hypersensitivity model of acute inflammation in mice. Contact hypersensitivity reactions to 2, 4-dinitrofluorobenzene were monitored by measuring ear thickness and cellular infiltration, both of which were observed to be maximal 24 h after ear challenge. A murine-specific phosphorothioate oligodeoxynucleotide and various control sequences were each passively encapsulated into 100-nm diameter large unilamellar vesicles composed of egg phosphatidylcholine and cholesterol. All formulations were administered as a single-bolus injection into the tail vein approximately 15 min after initiating ear inflammation. Oligodeoxynucleotide dose was varied from 5 to 50 mg/kg and the extent of inflammation was assessed 24 h later. Mice treated with free oligonucleotide, empty vesicles, or encapsulated control sequences showed no measurable effect on ear swelling or cellular infiltration compared with untreated controls. However, mice that received the active sequence encapsulated in lipid vesicles exhibited near baseline levels of ear thickness and leukocyte infiltration, similar to that observed in mice treated with a topical corticosteroid. These data demonstrate the utility of liposome-encapsulated intercellular adhesion molecule-1 antisense oligonucleotide as a novel anti-inflammatory therapeutic.

It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.

Phosphorothioate oligodeoxynucleotides produce splenomegaly and mononuclear cell infiltrates in multiple organs in mice after repeated i.v. administration. Several phosphorothioate oligodeoxynucleotides were studied to better understand the basis of immunostimulatory properties of these molecules in mice and to study the effects of chemically modified oligonucleotides. Chemical modifications examined included 5-methyl cytosine and 2'-methoxyethoxy substituents. Male mice (six per group) were treated with oligonucleotide concentrations of 0, 2, 10, or 50 mg/kg by i.v. injection every other day for 14 days. Immune stimulation was assessed 24 h after the last dose by measuring spleen weight, or histologic and immunohistochemical examination of liver and kidney. Immune stimulation was dose-dependent for the phosphorothioate oligodeoxynucleotides studied, but potency varied as a function of sequence. Results from this study reveal that there is a close correlation between the extent of splenomegaly and other evidence of immune stimulation, such as the severity of cell infiltrates in liver and kidney in mice. Immunohistochemical analysis indicated that cell infiltrates in liver and kidney were primarily mononuclear cells associated with increased expression of the endothelial-leukocyte cellular adhesion molecule intracellular adhesion molecule-1 and the cytokine interleukin-6. Immune stimulation was markedly decreased with oligonucleotides containing the 5-methyl cytosine and further decreased by 2'-methoxyethoxy modifications. Administration of these modified oligonucleotides to mice did not produce splenomegaly even at the 50-mg/kg dose, and only produced minimal cell infiltrates despite the presence of comparable or greater tissue oligonucleotide concentrations. Thus, chemical modifications appeared to increase the tolerability profile for these compounds that are representative of the second generation of antisense oligonucleotides.

Mice treated p.o. with 5% dextran sodium sulfate develop a mild to moderate colitis characterized by focal areas of inflammation and crypt abscesses. Immunohistological analysis of colons from dextran sodium sulfate-treated mice revealed an increased expression of intercellular adhesion molecule 1 (ICAM-1) and infiltration of lymphocyte function antigen 1-positive cells. A murine-specific antisense oligonucleotide, ISIS 3082, was used to determine the role of ICAM-1 expression in the development of colitis. Prophylactic treatment of dextran sodium sulfate-treated mice with ISIS 3082 reduced the clinical signs of colitis in a dose-dependent manner, with maximal effects occurring at a dose of 1 mg/kg/day. Reductions in ICAM-1 immunostaining and infiltrating leukocytes were observed in colons of animals treated with 1 mg/kg ISIS 3082. Scrambled control oligonucleotides failed to modify the course of the disease. The ICAM-1 oligonucleotide also diminished the clinical severity of colitis in mice with established colitis. The toxicity of ISIS 3082 was assessed in normal CD-1 mice by administering the oligonucleotide intravenously every other day for 2 weeks. At pharmacologically relevant doses of ISIS 3082 (1 and 10 mg/kg), there were no signs of toxicity with respect to body and organ weights, clinical chemistry or hematology. At a dose of oligonucleotide 20- to 100-fold greater than maximal pharmacological doses, the oligonucleotide produced an increase in liver and spleen weights; a mild chronic inflammation in liver, lung and lymph nodes; monocytosis and an elevation of serum liver transaminases. These data suggest that an antisense oligonucleotide that reduces ICAM-1 expression could be effective in the therapy of inflammatory bowel disease in humans and that such an oligonucleotide would be safe at pharmacologically relevant doses.