A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix.

Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

A Blood Drying Process for DNA Amplification.

The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid-phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano-porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin-resistant Staphylococcus aureus, Methicillin-Sensitive Staphylococcus aureus using loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.

Discovery of the first selective, small-molecule GFRα2/3 inhibitors through DNA-encoded library technology.

Studies have shown that disrupting the formation of the ligand-RET-GFRα complex could be an effective way of treating pain and itch. Compared to traditional high-throughput screens, DNA encoded libraries (DELs) have distinguished themselves as a powerful technology for hit identification in recent years. The present work demonstrates the use of DEL technology identifying compound 16 as the first GFRa2/GFRa3 small molecule inhibitor (0.1/0.2 µM respectively) selective over RET. This molecule represents an opportunity to advance the development of small-molecule inhibitors targeting the GFRα-RET interface for the treatment of pain and itch.

Molecular mechanisms of the virulence and efficacy of a highly virulent Vibrio anguillarum strain and its formalin-inactivated vaccine in rainbow trout.

In this study, we have isolated four strains of Vibrio anguillarum, revealing that they share the same serotype of O1, biochemical characteristics and virulence factor genes. However, there were differences in haemolytic activity among the bacterial strains; a strain with lower pathogenicity showed γ-haemolytic activity, whereas other virulent strains showed α-haemolytic activity on blood agar and higher empA gene expression in RTG-2 cell line. The most virulent strain was V. anguillarum RTBHR from diseased masu salmon (Oncorhynchus masou), which resulted in mortality of 100% and 93.3% when injected intraperitoneally at concentrations of 9 × 105 and 6.3 × 105 colony-forming units/fish in rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), respectively. A formalin-inactivated vaccine of V. anguillarum RTBHR induced a protective and specific immunity in rainbow trout as the vaccinated fish exhibited low cumulative mortality in a challenge test and a high specific antibody response in enzyme-linked immunosorbent assay at 8 weeks post-vaccination. The produced antibody was bound to bacterial proteins of 30-37 kDa in size. This adaptive immune response was detected as early as day 1, with quantitative polymerase chain reaction analysis revealing the upregulated expression of genes encoding for TCRα, T-bet, mIgM and sIgM in rainbow trout. This suggested that the vaccine induced T (probably a more dominant Th1 response) and B cell responses. In conclusion, the vaccine successfully protected fish from V. anguillarum infection by eliciting cellular and humoral immune responses.

Magnetic-Plasmonic Multimodular Hollow Nanoreactors for Compartmentalized Orthogonal Tandem Catalysis.

In tandem catalytic systems, controlling the reaction steps and side reactions is extremely challenging. Here, we demonstrate a nanoreactor platform comprising magnetic- and plasmonic-coupled catalytic modules that synchronizes reaction steps at unconnected neighboring reaction sites via decoupled nanolocalized energy harvested using distinct antennae reactors while minimizing the interconflicting effects. As was desired, the course of the reaction and product yields can be controlled by a convenient remote operation of alternating magnetic field (AMF) and near-infrared light (NIR). Following this strategy, a tandem reaction involving [Pd]-catalyzed Suzuki-Miyaura C-C cross-coupling and [Pt]-catalyzed aerobic alcohol oxidation enabled an excellent yield of cinnamaldehyde (ca. 95%) by overcoming the risk of side reactions. The customization scope for using different catalytic metals (Pt, Pd, Ru, and Rh) with in situ control over product release through remotely operable benign energy sources opens avenues for designing diverse catalytic schemes for targeted applications.

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

Effects of sulfated polysaccharides isolated from Codium fragile on inflammatory cytokine gene expression and Edwardsiella tarda infection in rockfish, Sebastes schlegelii.

Sulfated polysaccharides (SPs) derived from Codium fragile (sponge seaweed) can regulate cytokine expression in mammalian macrophages, NK cell lines and olive flounder head kidney primary cells in vitro. In this study, we found that SPs from C. fragile exhibited anti-bacterial activities against fish pathogenic bacteria including Streptococcus parauberis, Lactococcus garvieae, Aeromonas salmonicida and Edwardsiella tarda at a minimum inhibitory concentration of 2 mg/mL, but not against S. iniae or Vibrio anguillarum. Immunostimulatory effects of SPs from C. fragile on rockfish (Sebastes schlegelii) were evaluated by analyzing mRNA expression levels of inflammatory cytokines (interleukin (IL)-1ß, IL-8, IL-6 and tumor necrosis factor (TNF)-α) and anti-inflammatory cytokines (IL-10) both in vitro and in vivo. Results revealed that expression levels of all genes tested were upregulated in rockfish head kidney and spleen cells by SPs from C. fragile in a dose/time-dependent manner in vitro. By contrast, expression levels of these genes were significantly (p < 0.05) downregulated in the head kidney and spleen of rockfish in vivo at 1 and 3 days post intraperitoneal injection of SPs from C. fragile. In the liver, these genes were downregulated on day 1, but upregulated on day 3. Treatment with SPs downregulated the expression of these genes in spleen, but upregulated IL-10 gene expression in the intestine and liver. Meanwhile, when fish were fed with crude SPs for 4 weeks and challenged with E. tarda, infected fish started to die starting from 2 days after immune challenge. The cumulative mortality of the 0.1% group was significantly lower (p < 0.05) than that of the control group without feeding with SPs. Expression levels of IL-1ß and IL-6 genes were significantly (p < 0.05) upregulated in head kidney of the 0.5% group on day 1 while IL-1ß gene expression was downregulated on day 3 in the liver. These results indicate that SPs from C. fragile can regulate the immune gene expression in rockfish and that a diet containing 0.1% crude SPs can reduce the mortality of rockfish caused by E. tarda infection.

Transcriptome profiling in head kidney of rainbow trout (Oncorhynchus mykiss) after infection with the low-virulent Nagano genotype of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) causes clinical diseases and mortality in a wide variety of salmonid species. Here, we studied transcriptional responses in rainbow trout infected by the IHNV-Nagano strain isolated in Korea. RNA-seq-based transcriptome analysis of head kidney tissues cataloged differentially expressed genes. Enrichment analysis of gene ontology annotations was performed, and a total of fifteen biological process terms were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes pathway analysis, pathogen recognition receptor (PRR) signaling pathways such as the retinoic-acid-inducible gene-I-like receptor signaling pathway and the Toll-like receptor signaling pathway were identified at all time points. The nucleotide-binding oligomerization-domain-like receptor signaling pathway and cytosolic DNA-sensing pathway were identified at days 1 and 3. Protein-protein interaction network and centrality analyses revealed that the immune system, signaling molecules, and interaction pathways were upregulated at days 1 and 3, with the highest centrality of tumor necrosis factor. Cancer, cellular community, and endocrine system pathways were downregulated, with the highest centrality of fibronectin 1 at day 5. STAT1 was upregulated from days 1 to 5 with a high centrality. The reproducibility and repeatability of the transcriptome analysis were validated by RT-qPCR. IHNV-Nagano infection dynamically changed the transcriptome profiles in the head kidney of rainbow trout and induced a defense mechanism by regulating the immune and inflammatory pathways through PRR signaling at an early stage. Downregulated pathways involved in extracellular matrix formation and focal adhesion at day 5 indicated the possible failure of wound healing, which is important in the pathogenesis of IHNV infection.

Magnetothermia-Induced Catalytic Hollow Nanoreactor for Bioorthogonal Organic Synthesis in Living Cells.

Nanoreactors, in which the reactions are remotely controlled by magnetic fields, are potentially valuable in bioorthogonal chemistry for future applications. Here, we develop a silica-confined magnetothermia-induced nanoreactor (MAG-NER) by selectively growing Pd nanocrystals on a preinstalled iron-oxide core inside a hollow silica nanoshell. The growth is achieved by magnetic induction. The interfacial catalytic site is activated by stimulating localized magnetothermia, and nanocompartmentalization is realized by the size-selective porous silica. Therefore, MAG-NER can be conveniently used in complex biomedia and can even be internalized to living cells, realizing an on-demand, high-performance intramolecular carbocyclization reaction by remote operation without compromising the cell viability. This work opens avenues for the design of advanced nanoreactors that complement and augment the existing bioorthogonal chemical tools.

Au/Pt-Egg-in-Nest Nanomotor for Glucose-Powered Catalytic Motion and Enhanced Molecular Transport to Living Cells.

Nanostructures converting chemical energy to mechanical work by using benign metabolic fuels, have huge implications in biomedical science. Here, we introduce Au/Pt-based Janus nanostructures, resembling to "egg-in-nest" morphology (Au/Pt-ENs), showing enhanced motion as a result of dual enzyme-relay-like catalytic cascade in physiological biomedia, and in turn showing molecular-laden transport to living cells. We developed dynamic-casting approach using silica yolk-shell nanoreactors: first, to install a large Au-seed fixing the silica-yolk aside while providing the anisotropically confined concave hollow nanospace to grow curved Pt-dendritic networks. Owing to the intimately interfaced Au and Pt catalytic sites integrated in a unique anisotropic nest-like morphology, Au/Pt-ENs exhibited high diffusion rates and displacements as the result of glucose-converted oxygen concentration gradient. High diffusiophoresis in cell culture media increased the nanomotor-membrane interaction events, in turn facilitated the cell internalization. In addition, the porous network of Au/Pt-ENs facilitated the drug-molecule cargo loading and delivery to the living cells.

Silica Jar-with-Lid as Chemo-Enzymatic Nano-Compartment for Enantioselective Synthesis inside Living Cells.

Nanodevices, harvesting the power of synthetic catalysts and enzymes to perform enantioselective synthesis inside cell, have never been reported. Here, we synthesized round bottom jar-like silica nanostructures (SiJARs) with a chemo-responsive metal-silicate lid. This was isolated as an intermediate structure during highly controlled solid-state nanocrystal-conversion at the arc-section of silica shell. Different catalytic noble metals (Pt, Pd, Ru) were selectively modified on the lid-section through galvanic reactions. And, lid aperture-opening was regulated by mild acidic conditions or intracellular environment which accommodated the metal nanocrystals and enzymes, and in turn created an open-mouth nanoreactor. Distinct from the free enzymes, SiJARs performed asymmetric aldol reactions with high activity and enantioselectivity (yield >99 %, ee=95 %) and also functioned as the artificial catalytic organelles inside living cells. This work bridges the enormous potential of sophisticated nanocrystal-conversion chemistry and advanced platforms for new-to-nature catalysis.

Small noncoding RNAs in FSHD2 muscle cells reveal both DUX4- and SMCHD1-specific signatures.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by insufficient epigenetic repression of D4Z4 macrosatellite repeat where DUX4, an FSHD causing gene is embedded. There are two forms of FSHD, FSHD1 with contraction of D4Z4 repeat and FSHD2 with chromatin compaction defects mostly due to SMCHD1 mutation. Previous reports showed DUX4-induced gene expression changes as well as changes in microRNA expression in FSHD muscle cells. However, a genome wide analysis of small noncoding RNAs that might be regulated by DUX4 or by mutations in SMCHD1 has not been reported yet. Here, we identified several types of small noncoding RNAs including known microRNAs that are differentially expressed in FSHD2 muscle cells compared to control. Although fewer small RNAs were differentially expressed during muscle differentiation in FSHD2 cells compared to controls, most of the known myogenic microRNAs, such as miR1, miR133a and miR206 were induced in both FSHD2 and control muscle cells during differentiation. Our small RNA sequencing data analysis also revealed both DUX4- and SMCHD1-specific changes in FSHD2 muscle cells. Six FSHD2 microRNAs were affected by DUX4 overexpression in control myoblasts, whereas increased expression of tRNAs and 5S rRNAs in FSHD2 muscle cells was largely recapitulated in SMCHD1-depleted control myoblasts. Altogether, our studies suggest that the small noncoding RNA transcriptome changes in FSHD2 might be different from those in FSHD1 and that these differences may provide new diagnostic and therapeutic tools specific to FSHD2.

Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis.

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103  CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A-layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin-killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post-vaccination, respectively, in challenge test.

Nanocatalosomes as Plasmonic Bilayer Shells with Interlayer Catalytic Nanospaces for Solar-Light-Induced Reactions.

Interest and challenges remain in designing and synthesizing catalysts with nature-like complexity at few-nm scale to harness unprecedented functionalities by using sustainable solar light. We introduce "nanocatalosomes"-a bio-inspired bilayer-vesicular design of nanoreactor with metallic bilayer shell-in-shell structure, having numerous controllable confined cavities within few-nm interlayer space, customizable with different noble metals. The intershell-confined plasmonically coupled hot-nanospaces within the few-nm cavities play a pivotal role in harnessing catalytic effects for various organic transformations, as demonstrated by "acceptorless dehydrogenation", "Suzuki-Miyaura cross-coupling" and "alkynyl annulation" affording clean conversions and turnover frequencies (TOFs) at least one order of magnitude higher than state-of-the-art Au-nanorod-based plasmonic catalysts. This work paves the way towards next-generation nanoreactors for chemical transformations with solar energy.

Thermal Properties of Schottky Barrier Diode on AlGaN/GaN Heterostructures on Chemical Vapor Deposition Diamond.

High electron mobility transistors (HEMTs) and Schottky barrier diodes (SBDs) based on AlGaN/GaN heterostructure have been widely studied for high-frequency and/or high-power application. Widely distributed substrates for the high performance of RF applications are presently AlGaN/GaN on SiC, and those for high power performance are AlGaN/GaN on Si. Because the thermal conductivity of CVD diamond substrates is as high as 12 W/cm · K, devices on AlGaN/GaN on CVD diamond are one of the excellent alternatives for power and RF applications. In comparison, the thermal conductivity of AlGaN/GaN on SiC is 4.9 W/cm K, and that of AlGaN/GaN on Si is 1.3 W/cm · K. In this work, we report the fabrication of SBD devices with 163.8 mm Schottky channel length. We also compared the thermal properties of the fabricated large scale SBD devices on different substrates.

DC Characteristics of AlGaN/GaN High-Electron Mobility Transistor with a Bottom Plate Connected to Source-Bridged Field Plate Structure.

We investigate DC characteristics of AlGaN/GaN high-electron mobility transistors by using a source-bridged field plate and additional bottom plate (BP) structure. The analysis of experimental data was performed with a two-dimensional simulator. Source connected BP structure stabilized threshold voltage and transconductance regardless of various drain voltages. The effect of BP location was also analyzed, which had optimal DC values because of the dependence of breakdown voltage and drain current of the device on BP position between gate and drain. Finally, the optimum distance of 0.8 µm from drain side gate head edge to BP was achieved for optimum DC characteristics and the highest breakdown voltage of 341 V.

Non-Linear Cellular Dielectrophoretic Behavior Characterization Using Dielectrophoretic Tweezers-Based Force Spectroscopy inside a Microfluidic Device.

Characterization of cellular dielectrophoretic (DEP) behaviors, when cells are exposed to an alternating current (AC) electric field of varying frequency, is fundamentally important to many applications using dielectrophoresis. However, to date, that characterization has been performed with monotonically increasing or decreasing frequency, not with successive increases and decreases, even though cells might behave differently with those frequency modulations due to the nonlinear cellular electrodynamic responses reported in previous works. In this report, we present a method to trace the behaviors of numerous cells simultaneously at the single-cell level in a simple, robust manner using dielectrophoretic tweezers-based force spectroscopy. Using this method, the behaviors of more than 150 cells were traced in a single environment at the same time, while a modulated DEP force acted upon them, resulting in characterization of nonlinear DEP cellular behaviors and generation of different cross-over frequencies in living cells by modulating the DEP force. This study demonstrated that living cells can have non-linear di-polarized responses depending on the modulation direction of the applied frequency as well as providing a simple and reliable platform from which to measure a cellular cross-over frequency and characterize its nonlinear property.

DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD.