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SARS-CoV2 Research Blog


26May 20

05.26.20 progress update

We have now performed experiments in HT29 and HepG2 cells to test mitigation/synthetic lethality between remdesivir and drugs that impact mitochondrial function, following on our RNA-seq analysis showing reduced mitochondrial gene expression after remdesivir treatment (reproduced below:) . Our cell line survival results are shown below: In both cell lines, adenosine and syrosingopine rescue toxicity and ursodeoxycholic acid exacerbates toxicity. These effects are more pronounced in HT29. Adenosine is easy to explain-- it is a direct competitor of remdesivir (which is a modified adenosine derivative). So, under the hypothesis that remdesivir toxicity is caused by inhibition of mitochondrial RNA polymerase, the more wild-type adenosine available to the cell, the less RNA polymerase has to encounter remdesivir. The results for Ursodeoxycholic acid and syrosingopine are more interesting, and these drugs can be posited as antagonistic at the level of mitochondrial function. Ursodeoxycholic acid has been shown to improve mitochondrial function, while syrosingopine has been shown to inhibit mitochondrial function through blocking lactate export and thus causing feedback inhibition of lactate dehydrogenase and inhibition of mitochondrial electron…

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08May 20

05.08.20 Update

We are working hard on collecting more CRISPR screening data and optimizing our RdRp and SARS-CoV-2 entry assays. Meanwhile, our lab's COVID-19 work was written up here:

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30Apr 20

04.30.20 progress update

While analysis of our CRISPR screens and initial RdRP experiments are still ongoing, we are making progress analyzing and following up on the RNA-seq data on remdesivir and HCQ host response. HCQ: RNA-seq had identified upregulation of cholesterol metabolism genes as a consistent effect of HCQ treatment: So we asked whether HCQ impairs cholesterol uptake due to its role as a endosome to lysosome trafficking inhibitor and found a strong HCQ-dependent reduction in LDL uptake: We then asked whether this might lead to synthetic lethality with other drugs that impair cholesterol production. We identified robust synthetic lethality between HCQ and Simvastatin: Next, we asked whether this HCQ impairment of cholesterol and this synthetic lethality with Simvastatin might be mitigated by cholesterol intermediates. We found that mevalonate, a cholesterol intermediate, could completely mitigate HCQ toxicity in HepG2 and HT29 cells, completely mitigate HCQ+Simvastatin lethality in HT29 and partially mitigate it in HepG2. CoQ10, a similar intermediate in cholesterol-related biosynthetic pathways, had similar efficacy in mitigation of HCQ toxicity but was less effective at mitigating combined HCQ+Simvastatin…

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21Apr 20

Week 4 progress update

This week, we have continued analyzing our RNA-seq data, we have completed and sequenced our CRISPR screens, and we have cloned and are performing initial RdRP experiments. Stay tuned for updates on the CRISPR screen in the next few days. Regarding RNA-seq, we are working on how best to develop coherent GSEA analysis (Fig. 1a). While we move forward in this area, our most tangible progress has been in evaluating the RNA-seq finding that HCQ impairs cholesterol uptake in our cell lines. We have performed fluorescent LDL uptake assays, showing significantly reduced LDL uptake upon HCQ dosing by flow cytometry (Fig. 1b) and an LDL endosome retention phenotype by fluorescent microscopy (Fig. 1c) that support the mechanistic hypothesis that HCQ impairment of endosome to lysosome processing underlies impairs LDL uptake.  Because statins are widely prescribed drugs that impair cholesterol metabolism, we tested whether there is synthetic lethality between HCQ and Simvastatin in our cell lines as a result of excessive cholesterol starvation. We find reproducible synthetic lethality between HCQ and Simvastatin in several cell lines…

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13Apr 20

Week 3 progress update

Here’s an update on our progress this week: RNA-seq We are actively analyzing and following up on the RNA-seq data we collected last week. We have uploaded the raw data and some processed files run through gene (DESeq2) and pathway (GSEA) analysis tools. While we are actively analyzing these data, we are also already pursuing promising leads.Among these leads, the RNA-seq gave clear evidence from all 4 cell lines that HCQ activates cholesterol metabolism genes (see figure below), which is a sign that HCQ is blocking the ability of cells to uptake cholesterol. This phenotype fits with the known role of HCQ in preventing endosome-lysosome trafficking since cholesterol is taken into cells through LDLRs, which are processed through the endosome-lysosome pathway. To test whether HCQ does indeed block LDL uptake, we fed fluorescently labeled cholesterol to HepG2 and HT29 cells in the presence or absence of HCQ. In both cell lines, we see robust and significant decrease in LDL uptake (see figure below). We are currently asking whether this impairment in cholesterol uptake/processing contributes to…

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13Apr 20

Week 3 Update: Establishing a scalable mammalian cell fluorescence-based assay for SARS-CoV2 RdRP activity

**Note: We are currently searching for SARS-CoV2 genomic RNA or cDNA for use in our project! Our order from ATCC has been backordered until October and we are looking for an alternative. Please reach out to our lab if you may be able to help ( Background: SARS-CoV2 is a positive strand RNA virus with a large, 30 kb genome. The first two-thirds of the viral RNA (ORF1ab) is directly translated by the host upon viral infection of a cell to create one giant polyprotein. This polyprotein is posttranslationally cleaved to create 16 non-structural proteins. Many of these proteins are involved in replication, such as nsp 12, the RNA dependant RNA polymerase (RdRP) enzyme.1  The last third of the genome (ORFs 2-10) encodes for the SARS-CoV2 structural proteins. These ORFs are not directly translated by ribosomes upon infection. Rather, they are first transcribed by RdRP into negative strand subgenomic mRNAs (sgmRNAs), which are transcribed again into positive strand mRNAs.1 This process allows each gene to position itself at the 5’ end of the mRNA to…

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04Apr 20

Week 2 progress update

Fewer than two weeks after starting our experiments aimed at assessing genetic pathways mediating host toxicity of the SARS-CoV2 drugs remdesivir and hydroxychloroquine (HCQ), we already have our first experiment finished. Here’s an update on our progress this week: RNA-seq Minsun collected RNA from our 4 cell lines given doses of remdesivir and HCQ that we determined not to yield cell death within 8-24 hours (our two RNA-seq harvest timepoints) but high enough to affect cell physiology and gene expression (as determined by their toxicity starting 48 hours after dosing). RNA yields (listed in 033020_Rem_HCQ_RNAseq_experiment) revealed lower RNA concentrations in many drug treatment conditions, especially after 24 hours, suggesting that the doses we gave did affect cell physiology. Whether we hit the “Goldilocks” zone of a high enough dose to measure gene expression changes but not so high to induce massive pro-apoptotic gene expression remains to be seen, so we may have to repeat some conditions.Minsun performed Lexogen Quantseq 3’ RNA-seq prep (this is a robust kit that we have used successfully in the past)…

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01Apr 20

Optimizing drug dosing

We finished our first titration of remdesivir and HCQ, and, as is so typical, the conclusion was that we need to do it again. That’s not the full story though. We did determine optimal doses for our RNA-seq experiment, for which we have now already harvested cells, and we narrowed down the dose range for our CRISPR screen. To recap, we had two goals for our drug titration: 1.       RNA-seq: Determine a dose of remdesivir and HCQ that should NOT yield cell death within 8-24 hours (our two RNA-seq harvest timepoints) but should be high enough to affect cell physiology and gene expression. 2.       CRISPR screen: Determine a dose of remdesivir and HCQ that leads to 50% cell survival within a 4-6 day time window. For the RNA-seq dose, we used information from visual inspection of cells one and two days after treatment as well as flow cytometric live/dead analysis two days after treatment. Our chosen doses are noted in the 032520_drugtitration_experiment_0330summary and 033020_Rem_HCQ_RNAseq_experiment files. Note in the RNA-seq…

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30Mar 20

A role for genetic susceptibility in proposed controlled human vaccine challenge studies

A recent article ( has suggested the intriguing and ethically charged possibility of testing the efficacy of candidate SARS-CoV2 vaccines on healthy volunteers. The authors suggest testing candidate vaccines for efficacy by dosing the vaccines to healthy volunteers, infecting those volunteers with SARS-CoV2, and then closely monitoring signs of viral infection (while ensuring top-notch care during the testing period). They suggest that such studies, which would occur after Phase 1/2 safety and dosing trials and would be followed by an expanded placebo-controlled field trial, could subtract months from the vaccine development process. It is widely believed that the COVID-19 pandemic will not be quenched until the widespread rollout of effective vaccines. Due to the potentially enormous public health benefit of shaving months off of the vaccine testing process without added cost, I think that this idea deserves serious attention. It also deserves serious scrutiny due to the obvious downsides of societally encouraging healthy volunteers to put their health at risk for these studies. The merits and drawbacks of this approach should be considered through open…

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28Mar 20

Experiment set up: measuring CC50 for Remdesivir (GS-5734) and Hydroxychloroquine (HCQ)

Plate 8000 cells/well (2.5*10^4/cm2) of each cell line in 70 uL/well media in a 96 well format (2 x 48 wells per cell line—4 replicates each x 12 doses x 2 drugs). Make a mastermix for 48*1.1 wells and aliquot to wells using multichannel pipettor to be as even as possible. Add 10 uL/well of a mix of OptiMEM + drug (prepared in 96-well round bottom plate to use multi-channel pipettor Monitor cells the next day (24 hrs after plating and drug addition) and make notes of cell death trends. Do not change media. The next day (48 hrs after plating and drug addition), perform flow cytometric analysis on 2 of the 4 wells/cell line and drug condition (protocol below). Feed the remaining 2 of 4 wells/cell line and drug condition with the same drug dose using 190 uL media volume per well + 10 uL OptiMEM + drug—increase drug amount proportionally. Perform flow cytometric analysis 4-5 days post-initial drug addition on this second set of wells. Flow cytometric analysis There is no need to…

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28Mar 20

Drug titration: day 2 timepoint

We set up a titration testing 11 concentrations each of remdesivir and hydroxychloroquine (HCQ) each on 4 cell lines (HepG2 and PLC/PRF/5 liver lines, HCT116 and HT29 intestinal lines). Our primary goal is to find a CC50 concentration, defined by us as a drug concentration for which there are 50% as many surviving cells after a 4-6 day period in the presence of each drug. Note that this is not necessarily the standard way CC50 is defined, but it best enables a CRISPR screen to determine host genetic dependencies for drug toxicity. To be more specific, this titration would give us the dose needed to perform a genome-wide CRISPR screen where we determine whether the knockout of any gene induces more or less cell survival over a 4-6 drug treatment period. This is the simplest CRISPR screening protocol since it requires relatively little drug feeding (re-feeding every 2-3 days, this means two doses of the drug) and doesn’t require any cell sorting, as we will simply collect genomic DNA from surviving cells after the treatment…

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28Mar 20

Week 1 update: getting started

A frantic week kicked off on Sunday, March 22 when we had our first Google Hangouts group call pitching and brainstorming ideas. In the week that has followed, our lab has accomplished a dizzying array of tasks of all kinds to get projects off the ground, culminating in our first data! It has taken a truly amazing amount of persistence and cooperation to get to where we are now. Here are some notes that attest to the wide range of tasks that needed to be accomplished even just to get started: · We started a group Slack account on Monday and a lab Twitter account on Friday. Already, Slack has now become an invaluable way to communicate as a group, and we hope Twitter will serve likewise for communicating with the broader community. · We ordered remdesivir (MedChemExpress), hydroxychloroquine (Selleck), an RNA-seq kit (Lexogen), two batches of genome-wide CRISPR lentiviral library (Addgene), and Illumina Nextseq kits, all while the lab was officially closed. We tried to order remdesivir from Selleck but they were out until at…

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28Mar 20

Welcome to Our Research Blog

Like so many people around the world, the 12 members of our lab have been following the global spread of the SARS-CoV2 coronavirus pandemic and wondering whether we can help out in any way. None of us are doctors. The science we think about on a daily basis (using high-throughput genomic techniques such as CRISPR screening to understand and develop therapies for genetic disease) doesn’t concern infectious disease or the body’s responses to it. Is there something, however modest, that we can do that may help others figure out how best to treat COVID-19 patients? To be honest, we’re not sure. But we have come up with a few areas where we think the tools of genomics and gene editing might provide insight that could come in handy to those on the front lines treating COVID-19 patients. We have begun efforts in a few different areas where we think the rapid collection of genomic datasets may have a non-zero chance of informing the medical community. Specifically, we see a possible area of need in understanding…

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Project Updates:

4/05/20 RNA-seq data for remdesivir and HCQ effects on liver and intestinal cell lines (200404-covid19-toxicity). This zip folder contains raw counts data (0320_Sherwood_Nextseq.counts), normalized log counts (0320_Sherwood_Nextseq.vsd), DESeq2 differential gene expression (0320_Sherwood_Nextseq-DESeq2 folder), and GSEA analysis for each cell line and drug (0320_Sherwood_Nextseq-GSEA folder) 

4/07/20 Final summary files for remdesivir and HCQ concentrations (033120_HCQ_HepG2_HT29_titration_v2, 040620_drugtitration_d6_d7FACS_results,  033020_Remdesivir_titration_v2,  0420_Remdesivir_HCQ_concentration_summary)

3/31/20 Second titration experiments for remdesivir and HCQ (033020_Remdesivir_titration_v2, 033120_HCQ_HepG2_HT29_titration_v2)

3/30/20 ongoing RNA-seq experiment for remdesivir and HCQ effects on liver and intestinal gene expression (033020_Rem_HCQ_RNAseq_experiment)

3/30/20 Flow cytometry results on remdesivir and HCQ drug titration 5 days post-treatment (032520_drugtitration_0330d5FACS_results)

3/30/20 summary of remdesivir and HCQ drug titration experiment observations 1, 2, and 4 days post-treatment (032520_drugtitration_experiment_0330summary)

3/27/20 Flow cytometry results on remdesivir and HCQ drug titration 2 days post-treatment (032520_drugtitration_0327d2FACS_results)


Project proposal: Understanding and mitigating toxicity of COVID-19 drugs through genomics

Twitter: @SherwoodLab

Aim 1: Measure changes to gene expression using RNA-seq from remdesivir and hydroxychloroquine treatment
Aim 2: Perform genome-wide CRISPR screening to determine genes whose knockout increases or decreases remdesivir and hydroxychloroquine cytotoxicity.
Project Overview:
The rapid spread of the SARS-CoV2 virus presents an unprecedented challenge to control the morbidity and mortality of a disease without the methodical process of drug discovery and testing. As such, near-term medical interventions focus on repurposing existing drugs as the 12-18-month vaccine development proceeds. The two current front-line treatments that have emerged are the Ebola RNA-dependent RNA polymerase inhibitor remdesivir1 and the related anti-malarial drugs chloroquine and hydroxychloroquine2 . There is evidence from in vitro studies that these drugs are effective at inhibiting SARS-CoV23 , clinical case studies have begun to emerge that tentatively suggest some clinical efficacy, and clinical trials are actively enrolling patients throughout the world4,5
However, there are toxicity concerns for both drugs, and the interactions between these drugs and human proteins that mediate toxicity are not known. While remdesivir has low reported toxicity in vitro and in monkeys1 , early COVID-19 case reports show potential gastrointestinal toxicity6 and the lack of large-scale testing means that possible gene-drug interactions may arise as treatment is scaled up. Hydroxychloroquine has extensive usage but has known toxicity in an appreciable subset of patients7,8. Thus, for both drugs, there is a lack of mechanistic understanding of which human genes mediate cytotoxicity. Understanding such gene-drug interactions could improve treatment in two ways. First, this knowledge could offer possible targets for therapeutic intervention alongside drug treatment, whether by dosing with another drug or supplementing with a particular nutrient or vitamin. Second, it could be used to screen patient populations with preexisting conditions or genetic variants for which treatment with each drug is inadvisable. Such information is likely to synergize with ongoing genome association study efforts to correlate patient genetic profile with COVID-19 drug response9 . Therefore, we aim to use RNA-seq and genome-wide CRISPR screening to profile the genetics of remdesivir and hydroxychloroquine cytotoxicity. In Aim 1, we will profile the global gene expression response of four human intestinal and liver cell lines to each drug. In Aim 2, we will perform genome-wide CRISPR screening to identify genes whose knockout sensitizes or desensitizes cells to drug-induced cell death. We will mine these datasets for druggable gene and pathway targets and will test candidate drugs or nutrients we predict will improve tolerance to each drug. We will post all data online immediately as we collect it to aid the global effort to develop optimal treatment regimens for SARS-CoV2 infection… Full Text

Click the photo below to see our COVID-19 research updates on Twitter!


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