Angiopoietin-like 3 (ANGPTL3) inhibits endothelial lipase and lipoprotein lipase. Injection of antisense oligonucleotides targeting
ANGPTL3
messenger RNA effects a reduction of atherogenic ...lipoproteins in humans and mice and a slowing of progression of atherosclerosis in mice.
Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ-AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor ...neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here, we tested this hypothesis using AR113Q knockin and human bacterial artificial chromosome/clone (BAC) transgenic mice that express the full-length polyQ-AR and display androgen-dependent weakness, muscle atrophy, and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery but not the CNS after subcutaneous administration. Suppression of polyQ-AR in the periphery rescued deficits in muscle weight, fiber size, and grip strength, reversed changes in muscle gene expression, and extended the lifespan of mutant males. We conclude that polyQ-AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.
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•AR-targeted antisense oligonucleotides suppressed gene expression in mice•Subcutaneous delivery suppressed AR gene expression in the periphery but not the CNS•Subcutaneous administration rescued disease in two mouse models of SBMA•Peripherally expressed polyQ AR contributes to disease and is a therapeutic target
Spinal and bulbar muscular atrophy is a CAG repeat disorder characterized by degeneration of the neuromuscular system that is caused by the polyglutamine androgen receptor (polyQ AR). Lieberman et al. developed antisense oligonucleotides to suppress expression of the mutant protein. They show that peripheral administration of these compounds rescues disease in two mouse models, without altering expression within the CNS. Their findings demonstrate an important contribution of peripherally expressed polyQ AR to disease and highlight a new therapeutic approach.
Long noncoding RNAs (lncRNAs) have recently emerged as important players in diverse cellular processes. Among them, a large fraction of lncRNAs are localized within cell nucleus. And several of these ...nuclear-retained lncRNAs have been found to regulate key nuclear processes, which brings up the requirement of effective genetic tools to explore the functions of this "dark matter" inside the nucleus. While siRNAs and shRNAs are widely used tools in loss-of-function studies, their general efficiency in depleting nuclear-retained lncRNAs is limited, due to the fact that the RNAi machinery is located mainly in the cytoplasm of mammalian cells. Here, we describe the usage of chemically modified chimeric DNA antisense oligonucleotides (ASO) in effective knockdown of nuclear-retained lncRNAs, with a focus on the detailed workflow from the design and synthesis of ASOs, to in vitro and in vivo delivery methods.
Lymphocytes circulate in a quiescent (G0) state until they encounter specific antigens. In T cells, quiescence is programed by transcription factors of the forkhead box O (FOXO) and Krüppel-like ...factor (KLF) families. However, the transcription factors that regulate B cell quiescence are not known. KLF4 is a candidate tumor suppressor gene in B lymphocytes, and thus a likely candidate for regulating B cell homeostasis. Here, we show that RNA and protein expression of murine KLF4 decreases following B cell activation. Forced expression of KLF4 in proliferating B cell blasts causes a G1 cell cycle arrest. This effect requires the DNA binding and transactivation domains of KLF4 and correlates with changes in the expression of known KLF target genes. We present evidence that Klf4 is a target gene for FOXO transcription factors, which also suppress B cell proliferation. To determine the effect of KLF4 loss-of-function, we generated mice with B cell-specific deletion of the Klf4 gene. These mice exhibited normal B cell development and function with no evidence of a lowered activation threshold. Collectively, our findings indicate that KLF4 has growth-suppressive properties in B cells but might be redundant with other members of the KLF family in maintaining B cell quiescence.
Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease‐associated aberrant activity of the pathway is often due to misregulation ...of the equilibrium state. The transcription factor NF‐κB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IκB, until an external stimulus triggers IκB degradation through an IκB kinase‐dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady‐state regulation of the NF‐κB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF‐κB‐bound and ‐unbound IκB proteins that are critical for accurate computational predictions of steady‐state IκB protein levels and basal NF‐κB activity. Simulations reveal a homeostatic NF‐κB signaling module in which differential degradation rates of free and bound pools of IκB represent a novel cross‐regulation mechanism that imparts functional robustness to the signaling module.
β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 ...years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.
β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates ...iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.
Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that ...is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.
To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation ...antisense oligonucleotide (ASO).
Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels.
Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes.
Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.
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