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BackgroundMalignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ.MethodsVia lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis.ResultsWe used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects.ConclusiondCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

IntroductionFew of the 5000–8000 snakebites reported to poison control centers annually in the USA are attributed to coral snakes. This study describes Texas coral snake envenomations reported to the North American Snakebite Registry.MethodsAll Texas coral snake envenomation cases reported to the registry were identified for the period from January 1, 2015, through December 31, 2019. Data reviewed for this study included details regarding the snake encounter, patient demographics, signs and symptoms, treatment, and outcomes. Descriptive statistics were used to report results.ResultsTen men and four nonpregnant women reported coral snake bites. The median patient age was 15.5 (range 5–72 years). There were 12 upper extremity bites and two bites to the lower extremity. The most common symptoms reported were paresthesias and pain. All subjects had paresthesias, often described as an “electric” sensation. Seven patients described them as painful. The most common clinical findings were erythema and swelling. No patient developed tissue damage, hematotoxicity, rhabdomyolysis, hypotension, weakness, or respiratory symptoms. Thirteen subjects were treated with opioids. Six patients were treated with antiemetics: three prophylactically and two for opioid-induced nausea. One patient developed nausea and non-bloody, nonbilious emesis within 1 hour of the bite, prior to receiving opioids. No patients were treated with antivenom. Antibiotics were not administered to any patient, and no infections were reported.ConclusionsEnvenomations from M. tener in Southeast Texas are characterized by painful paresthesias. Mild swelling and erythema are common. Neurotoxicity necessitating antivenom or mechanical ventilation did not occur.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current clinical therapies and known causes of familial hypercholesterolemia. We show that Cold Shock Domain-Containing Protein E1 (CSDE1) enhances hepatic LDLR mRNA decay via its 3′ untranslated region to regulate atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens, we found 40 specific regulators of the LDLR left unidentified by observational human genetics. Among these, we show that CSDE1 regulates the LDLR at least as strongly as the mechanistically distinct pathways exploited by the best available clinical therapies: statins and PCSK9 inhibitors. Additionally, we show that hepatic gene silencing of Csde1 treats diet-induced dyslipidemia in mice better than that of Pcsk9. Our results reveal the therapeutic potential of manipulating a newly identified key factor in the post-transcriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. We anticipate that our approach of modelling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, will serve as a generalizable template for the identification of therapeutic targets in other human disease states. Competing Interest Statement P.N. reports investigator-initiated grant support from Amgen, Apple, and Boston Scientific, and personal fees from Apple, Blackstone Life Sciences, and Novartis, all unrelated to the present work. R.S.W. is an employee of Amgen, Inc. D.S. is the scientific cofounder, shareholder, and director of Tenaya Therapeutics, unrelated to the present work. K.M.S. has consulting agreements for the following companies involving cash and/or stock compensation: Black Diamond Therapeutics, BridGene Bioscences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Merck, Mitokinin, Petra Pharma, Revolution Medicines, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma). J.S.C. has received consulting fees from Gilde Healthcare and is an unpaid scientific advisor to Eko, both unrelated to this work. Footnotes * The current version reflects additional mechanistic details of CSDE1's action, in vivo validation of CSDE1 as a cholesterol regulating target in zebrafish and mice, and a therapeutic proof of principle of CSDE1 gene silencing for cholesterol lowering.